Confidencial 23/3/2007

UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL

FACULDADE DE FARMÁCIA

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS

UNIVERSIDADE DE ROUEN

FACULDADE DE MEDICINA E FARMÁCIA

UNIDADE DE NEUROPSICOFARMACOLOGIA EXPERIMENTAL

F.R.E. 2735 C.N.R.S – I.F.R.M.P. 23

Estudo de moléculas potencialmente antidepressivas e analgésicas de espécies de Hypericum nativas do Rio Grande do Sul

Alice Fialho Viana

PORTO ALEGRE, 2007

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UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL

FACULDADE DE FARMÁCIA

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS

UNIVERSIDADE DE ROUEN

FACULDADE DE MEDICINA E FARMÁCIA

UNIDADE DE NEUROPSICOFARMACOLOGIA EXPERIMENTAL

F.R.E. 2735 C.N.R.S – I.F.R.M.P. 23

Estudo de moléculas potencialmente antidepressivas e analgésicas de espécies de Hypericum nativas do Rio Grande do Sul

Tese apresentada por Alice Fialho Viana para obtenção do TÍTULO DE DOUTOR em CiênciasFarmacêuticas

Orientadores: Prof Dr Stela Maris K. Rates Dr Jean Claude do Rego Pr Jean Costentin

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Tese apresentada ao Programa de Pós-Graduação em Ciências Farmacêuticas, em nível de Doutorado da Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul e aprovada em 12.2.2007, pela Banca Examinadora constituída por:

Prof. Dr. Didier Vieau Université de Lille-I

Prof. Dr. Grace Gosmann Universidade Federal do Rio Grande do Sul

Prof. Dr. João Batista Calixto Universidade Federal de Santa Catarina

V614e Viana, Alice Fialho Estudo de moléculas potencialmente antidepressivas e analgésicas de espécies de hypericum nativas do Rio Grande do Sul / Alice Fialho Viana – Porto Alegre : UFRGS, 2007. - xix, 196 p.: il., gráf., tab.

Tese(doutorado). UFRGS. Faculdade de Farmácia. Programa de Pós- Graduação em Ciências Farmacêuticas; Universidade de Rouen. Faculdade de Medicina e Farmácia. Unidade de Neuropsicofarmacologia Experimental.

1. Hypericum caprifoliatum. 2. Hypericum polyanthemum. 3. Antidepressivos. 4. Analgésicos. 5. Farmacologia. I. Rates, Stela Maris Kunze. II. Rego, Jean Claude do. III. Costentin, Jean. IV. Título.

CDU: 615.322:582.824

Bibliotecária responsável: Margarida Maria Cordeiro Fonseca Ferreira, CRB 10/480

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APRESENTAÇÃO

Esta tese está inserida no projeto Planejamento e Síntese de Substâncias de Origem Natural e Derivados com Potencial Atividade Farmacológica aprovado pelo acordo CAPES/COFECUB - BRASIL / FRANÇA (Número CAPES 418/03; Ata Aprovação PPG- CF nº 9 de 3 de maio de 2001). O Projeto CAPES/COFECUB tem como objetivo a obtenção de moléculas bioativas, através de síntese orgânica e a partir da flora nativa do Rio Grande do Sul, centrando-se em dois gêneros bem representados no Estado: Ilex e Hypericum.

Especificamente, esta tese está ligada ao subprojeto Prospecção de moléculas potencialmente antidepressivas e analgésicas em espécies do gênero Hypericum nativas do Rio Grande do Sul, e foi realizada na modalidade de cotutela entre o Programa de Pós-Graduação em Ciências Farmacêuticas - UFRGS (PPG-CF), sob responsabilidade da Prof Dr Stela Maris Kuze Rates e a Unidade de Neuropsicofarmacologia Experimental da Faculdade de Medicina e Farmácia da Universidade de Rouen – F.R.E. 2735 C.N.R.S – I.F.R.M.P. 23, Rouen, França, sob a responsabilidade de Dr. Jean Claude do Rego e Prof. Dr. Jean Costentin.

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PRESENTATION

Ce travail a été réalisé dans le cadre d’une thèse en cotutelle entre le Programme de Post- Graduation en Sciences Pharmaceutiques de la Faculté de Pharmacie de l’Université Fédérale de Rio Grande do Sul (Porto Alegre, Brésil), sous la responsabilité de Prof Dr Stela Maris Kuze Rates et le Laboratoire de Neuropsychopharmacologie Expérimentale, CNRS FRE 2735, à la Faculté de Médecine et Pharmacie de l’Université de Rouen (France), sous la responsabilité de Dr. Jean Claude do Rego et Prof. Dr. Jean Costentin . Il a porté sur l’Etude de molécules potentiellement antidépressives et analgésiques issues d’espèces d’Hypericum natives du sud du Brésil, qui est inscrit pleinement dans notre projet commun, sur l’Etude et la synthèse de substances naturelles ainsi que de leurs dérivées ayant (possédant) des activités pharmacologiques potentielles, approuvé par l’accord CAPES/COFECUB - BRESIL/FRANCE (sous le numéro 418/03). L’objectif de ce projet est l’obtention, soit par synthèse organique ou à partir de la flore du Rio Grande do Sul, de molecules bioactives à partir d’étude des genres: Ilex et Hypericum.

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“I am among those who think that science has great beauty. A scientist in the laboratory is not only a technician: is also a child placed before natural phenomena which impress him like a fairy tale.” Marie Curie (1867 - 1934)

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AGRADECIMENTOS

À Stela minha muy querida orientadora/amiga e principal responsável pela concretização desta tese. Ela aceitou-me como sou e transformou-me no que sou. A Gil e Vera pela amizade e co-orientação não oficial. À Grace pelo apoio na minha seleção de doutorado e por todos os outros “pepinos” que precisou resolver como coordenadora brasileira do acordo CAPES/COFECUB. Ao Prof. Dr. João Batista Calixto e à Prof. Dr. Grace Gosmann pela contribuição dada ao aceitarem participarem da avaliação desta tese. À Terezinha minha mátria. Ao Paulo meu amado pai que sei que torceu imensamente por mim e que gostaria de ter participado muito mais deste processo. Keka, Gilda, Gusti, Léo, Andresa, Michele, Enio e Ana (“minha bolsista perfeita”) pelo auxílio e paciência nos experimentos. À Rose pelo auxílio após os experimentos e carinho com os animais. Às famílias Fialho e Viana, que por todos os cantos do Brasil me apoiaram mesmo sem saber direito o que eu estava fazendo. Às minhas irmãs, Elisa e Lela, ao meu irmão Dida, e à Nani minha ‘mãedrasta’, por agüentarem minha TPM constante e apoio nos momentos mais difíceis. Às miguis, Aline, Cachos, Eliza, Deisy, Janine, Raquel, Riani, minhas irmãs do coração, por tudo... que não posso comentar aqui... Aos estudantes estrangeiros na França, especialmente espanhóis e italianos, e os brasileiros, em particular Franciele, Karime, Christine, Clarisse, Wilson e Fabrício; o espírito latino levou um pouco de calor para Normandia. À CAPES por financiar minha bolsa de estudo.

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REMERCIMENTS

Monsieur Jean Claude do Rego, mon cher directeur de thèse en France, qui m’a apporté son aide pour affronter toute la bureaucratie française, m’a appris les mystères de la biochimie et plus important, m’a reçu avec toute sa sympathie, sa générosité. Il a fait preuve de patience et de compréhension tout au long de ma thèse. Ses compétences et sa rigueur scientifique ont été essentielles pour l’aboutissement de ce travail de thèse.

Monsieur Jean Costentin, qui m’a accepté dans son laboratoire, m’a fait confiance et a mis à ma disposition tous les moyens nécessaires à l’accomplissement de ce travail. Nos discussions de résultats avec lui ont été, pour moi, des cours de pharmacologie.

Madame Nathalie Dourmap, qui m’a apporté son aide précieuse pour l’utilisation du système d’HPLC et des conseils de voyage.

Monsieur Didier Vieau, Professeur à l’Université de Lille-I, qui malgré ses nombreuses occupations, a accepté d’être l’un des rapporteurs de ce travail. Je voudrais ici lui témoigner toute ma reconnaissance pour l’honneur qu’il m’accorde en acceptant d’effectuer ce long voyage pour prendre part à ce jury au Brésil.

Messieurs Bertrand Naudin et François Janin qui m’ont apporté leurs aides précieuses au cours de nombreuses expérimentations.

Elisabeth Chapelot ma “migui” française

Hervé de Vienne ..., mon voisin, premier ami français, enseignent de français et ski, future medicin

A tous les étudiants étrangers que j’ai rencontré lors de mon séjour en France, spécialement les Brésiliens : Fraciele Moura, Karime Hajar, Christine Ruta, Clarisse Riveira, Wilson Maia et Fabrício Pereira ; notre esprit latin a apporté un peu de chaleur en Normandie.

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SUMÁRIO

LISTA DE ABREVIATURAS...... 11

LISTA DE FIGURAS...... 12

RESUMO...... 14

ABSTRACT...... 16

RESUME...... 18

I PARTE : INTRODUÇÃO E REVISÃO BIBLIOGRÁFICA...... 21

1. INTRODUÇÃO...... 22

2. OBJETIVOS...... 24

2.2 OBJETIVOS ESPECÍFICOS...... 24

3. REVISÃO DA LITERATURA...... 25

3.1. TRANSTORNOS DEPRESSIVOS...... 25

3.1.1 Dados epidemiológicos ...... 27

3.1.2 Tratamentos...... 28

3.1.2.1. Antidepressivos Tricíclicos (ADT)...... 28

3.1.2.2. Inibidores de monoamino oxidases (IMAO)...... 29

3.1.2.3. Inibidores seletivos da recaptação de serotonina (ISRS)...... 30

3.1.2.4. Antidepressivos atípicos...... 30

3.1.3. Pacientes resistentes...... 33

3.2. BASES NEUROQUIMCAS DA DEPRESSÃO...... 34

3.2.1 Teoria monoaminérgica...... 34

3.2.2. Teoria neurotrófica...... 36

3.2.3. Eixo hipotálamo-pituitária-adrenal...... 39

3.3. O GÊNERO HYPERICUM)...... 45

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3.3.1 Dados farmacológicos do gênero Hypericum...... 46

3.4. REVISÃO DOS DADOS DE ESPÉCIES DE HYPERICUM NATIVAS DO RIO 57 GRANDE DO SUL...... 3.4.1 Dados químicos...... 57

3.3.2 Dados biológicos...... 59

II PARTE: RESULTADOS EXPERIMENTAIS...... 63

CAPÍTULO 1: ARTIGOS PUBLICADOS...... 64

Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae) The antidepressant like effect of Hypericum caprifoliatum Cham & Schlecht (Guttiferae) on forced swimming test results from an inhibition of neuronal monoamine uptake Hypericum caprifoliatum (Guttiferae) Cham. & Schltdl.: a species native to South Brazil with antidepressant like activity. CAPÍTULO 2: Effects of Hypericum caprifoliatum Cham. & Schltdt. (Guttiferae) 89 extract on serum and brain corticosterone levels...... CAPÍTULO 3: Study of antidepressant and antinociceptive activities of Hypericum 115 polyanthemum Klotzsch ex Reichardt...... CAPÍTULO 4: Effect of Hypericum caprifoliatum and Hypericum polyanthemum 146 cyclohexane extracts on monoamine and opioid receptor-stimulated [35S]GTPγS binding in rat brain membranes...... III PARTE: DISCUSSÃO, CONCLUSÕES E PERSPECTIVAS...... 167

REFERÊNCIAS...... 179

BIOGRAFIA...... 199

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LISTA DE ABREVIATURAS

APO apomorfina

BUP bupropiona

Fração com floroglucinóis do extrato ciclo- HC1 hexano de H. caprifoliatum

Extrato ciclo-hexano purificado das partes HCP aéreas de H. caprifoliatum

HP4 Uliginosina B

MOR morfina

NAL naloxona Extrato ciclo-hexano das partes aéreas de POL H. polyanthemum SAL solução salina (NaCl 0,9%)

SCH SCH 23390

SUL sulpirida

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LISTA DE FIGURAS

Figure 1. Monoaminergic, corticoid and immunological systems integration...... 43

Figure 2.1. The influence of stress and antidepressant treatment on CA3 pyramidal neurons in the hippocampus...... 95 Figure 2.2. Effect of acute or repeated treatment with imipramine, bupropion, H. caprifoliatum or saline on forced swimming test...... 100 Figure 2.3. Effect of acute treatment with imipramine, bupropion, H. caprifoliatum or saline on serum and cortical corticosterone levels...... 102 Figure 2.3. Effect of repeated treatment with imipramine, bupropion, H. caprifoliatum or saline on serum and cortical corticosterone levels...... 104 Figure 3.1. Molecules isolated from the lipophilic extracts of Southern Brazil Hypericum species...... 119 Figure 3.2. Anti-immobility effect of imipramine and H. polyanthemum in rat forced swimming test...... 127 Figure 3.3. Anti-immobility effect of imipramine and H. polyanthemum in mice forced swimming test...... 128 Figure 3.4. Anti-immobility effect of imipramine and uliginosin B in mice forced swimming test...... 128 Figure 3.5. Effect of SCH 23390) or sulpiride administration on the anti-immobility action of POL in mice forced swimming test...... 129 Figure 3.6. Effect of HP4 on [3H]-dopamine, [3H]-noradrenaline and [3H]-serotonin synaptosomal uptake...... 130 Figure 3.7. Effect of HP4 on [3H]-mazindol, [3H]-nisoxetine, [3H]-citalopram binding to dopamine, noradrenaline or serotonin transporters...... 131 Figure 3.8. Anti-immobility effect of acute and three days treatment with imipramine or H. polyanthemum in mice forced swimming test...... 132 Figure 3.9. Effect of acute per os treatment with imipramine, H. polyanthemum or saline on serum and cortical corticosterone levels...... 139 Figure 3.10. Effect of repeated treatment (3 days) with imipramine, H. polyanthemum or saline on serum and cortical corticosterone levels...... 135

Figura 3.11: Antinociceptive effect of morphine, benzopyrans HP1-3 and uliginosin B in the hot plate test...... 136 Figure 3.12. Effect of naloxone administration on the antinociceptive effect of HP4 and morphine in the hot plate test...... 137 Figure 3.13. Effect of HP4 on [3H]-naloxone binding to opioid receptors...... 137

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Figure 4.1: Effect of T2, T3 and T4 of H. caprifoliatum, H. polyanthemum or saline on rat forced swimming test...... 153 Figure 4.2: Effect of acute treatment with the phloroglucinols HC1 and HP4, or saline on mice forced swimming test...... 154 Figure 4.3: Effect of T1 with HCP, POL or saline on on agonist-stimulated [35S]GTPγS binding measured ex vivo...... 155 Figure 4.4: Effect of T2 with HCP, POL or saline on agonist-stimulated [35S]GTPγS binding measured ex vivo...... 156 Figure 4.5: Effect of T3 with HCP, POL or saline on agonist-stimulated [35S]GTPγS binding measured ex vivo...... 157 Figure 4.6: Effect of T4 with HCP, POL or saline on agonist-stimulated [35S]GTPγS binding measured ex vivo...... 158 Figure 4.7: Concentration-response effect of HC1 and HP4 on [35S]GTPγS binding...... 160 Figure 2: Phylogenetic tree of transporter proteins...... 173 Figure 3: H. caprifoliatum, H. polyanthemum extractio scheme and pharmacological activities resume...... 175

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RESUMO

Estudo de moléculas potencialmente antidepressivas e analgésicas de espécies de Hypericum nativas do Rio Grande do Sul

O objetivo deste trabalho foi estudar a ação antidepressiva e analgésica de extratos ciclo-hexano de Hypericum caprifoliatum (HCP) e Hypericum polyanthemum (POL) e seus principais constituintes químicos, os derivados floroglucinol, HC1 e uliginosina (HP4), respectivamente. HCP, POL, HC1 e HP4 reduziram significativamente a imobilidade de ratos e camundongos no teste de natação forçada (FST), indicando potencial atividade antidepressiva. Os efeitos de HCP e POL no FST foram prevenidos pelo pré-tratamento com

SCH23390 e sulpirida, antagonistas de receptores D1 e D2 respectivamente. Os extratos, HC1 e HP4 inibiram de modo dose-dependente a recaptação sinaptossomal de dopamina ([3H]-DA), noradrenalina ([3H]-NA) e serotonina ([3H]-5HT), com uma maior potência para a recaptação de DA. Entretanto, este efeito não parece ser dependente de uma ação direta das substâncias sobre o transportador de monoaminas, uma vez que diferentes concentrações de HC1 e HP4 não afetaram a ligação de [3H]-mazindol, [3H]-nisoxetina e [3H]- citalopram aos sítios de recaptação de DA, NA e 5-HT, respectivamente. Para avaliação do efeito dos extratos sobre os receptores de DA, NA, 5-HT e opióides, foi utilizada a técnica de ligação de [35S]-GTPγS estimulada por agonistas. O tratamento agudo de ratos com HCP ou POL aumentou significativamente a ligação de [35S]-GTPγS estimulada por DA, NA e 5-HT, enquanto 5 dias de tratamento diminuiram esta ligação. Nenhum dos regimes de tratamento afetou a ligação estimulada por DAMGO, agonista opióide. Estes resultados demonstram que os tratamentos com HCP e POL resultam em alterações na transmissão monoaminérgica. Porém estas não se devem a efeitos diretos de HC1 e HP4 sobre os receptores, pois a incubação com HC1 ou HP4 não alterou a ligação de [35S]-GTPγS em nenhuma preparação estudada. Além do estudo sobre o sistema monoaminérgico, foi verificada a ação de HCP e POL sobre o eixo HPA através da medida dos níveis séricos e plasmáticos de corticosterona em camundongos submetidos ou não ao estresse da natação forçada. Três dias de tratamento com HCP e POL reduziram significativamente o aumento do nível cortical de corticosterona induzido pelo nado forçado, sem afetar o nível sérico, evidenciando que o

xxi Confidencial 23/3/2007 tratamento repetido com os extratos afeta repostas hormonais ao estresse. Em relação à atividade analgésica, HCP e POL apresentaram efeito antinociceptivo na placa quente, o qual foi prevenido pelo pré-tratamento com naloxona. Entretanto, HC1 e HP4 não inibiram a 3 ligação da [ H]-naloxona, o efeito antinociceptivo de HP4 não foi prevenido pela naloxona, e o tratamento com os extratos, assim como a incubação direta HC1 ou HP4 não modificou a ligação de [35S]-GTPγS ao receptor opióide. Os resultados apresentados confirmam que HCP e POL têm potencial atividade antidepressiva e analgésica, provavelmente com um mecanismo de ação diferente dos fármacos atuais, sendo os derivados floroglucinol os prováveis responsáveis por estes efeitos.

Palavras-chave: Hypericum caprifoliatum, Hypericum polyanthemum, antidepressivo, analgésico, teste de natação froçada, placa-quente, recaptação sinaptossomal, ligação aos transportadores, [35S]-GTPγS, corticosterona.

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ABSTRACT

Prospective of molecules with antidepressant and analgesic activities from Hypericum species native to Rio Grande do Sul.

This work aimed to study the antidepressant and analgesic effect of Hypericum caprifoliatum (HCP) and H. polyanthemum (POL) cyclohexane extracts, as well as the substances and mechanisms involved on their activities. HCP, POL and their phloroglucinol derivatives (HC1 and HP4, respectively) significantly reduced rats and mice immobility time in the forced swimming test (FST), indicating potential antidepressant effect. The extracts showed some selectivity for activating the dopaminergic system. The antidepressant-like effect of HCP and POL was inhibited by D1 and D2 antagonists; and their extracts, HC1 and HP4, inhibited dopamine ([3H]-DA), noradrenaline ([3H]-NA) and serotonin ([3H]-5HT) uptake, more potently of dopamine. However, this effect is not related to the binding to monoamine transporters, since they did not affect the binding of [3H]-mazindol, [3H]-nisoxetine and [3H]- citalopram to DA, NA and 5-HT transporters, respectively. In order to investigate the functional role of DA, NA, 5-HT and opioid receptors in the effects of HCP and POL, we evaluated the extracts effect on monoamine and opioid receptor-stimulated [35S]-GTPγS binding. We demonstrated that acute or 3 treatments within 24h increases [35S]-GTPγS binding to DA, NA and 5-HT sites, while after 5 days of treatment the binding is reduced. Non treatment regimen affected binding to opioid sites. Hence, the results demonstrate that the treatment with HCP and POL induce alterations in monoaminergic transmission. As seen at the binding to monoamine transporters, these effects are not due to HC1 or HP4 interaction with the receptors, since direct incubation of HC1 and HP4 did no affect [35S]-GTPγS binding to membranes. Besides studying HCP and POL effects on the monoaminergic system we investigated their activity on other system related to depression, the hypothalamus-pituitary- adrenal axis. We demonstrated that 3 days treatment with the extract reduce cortical corticosterone but not serum levels increased by FST. Regarding to the analgesic activity, HCP and POL antinociceptive effect in the hot plate were prevented by naloxone. However, HC1 and HP4 did not inhibited [3H]-naloxone binding to opioid receptors. In conclusion, HCP and POL have potencial antidepressant and analgesic activities and the phloroglucinol

xxiii Confidencial 23/3/2007 derivatives are very likely to be the substances responsible for them. Even though the mechanism of action involves the monoaminergic system, the manner by which the extracts influence this system is different from classical antidepressants.

Keywords: Hypericum caprifoliatum, Hypericum polyanthemum, antidepressant, nociception, forced swimming test, hot-plate test, synaptosomal uptake, transporters binding, [35S]-GTPγS, corticosterone level.

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RESUME

Etudes de molécules potentiellement antidépressives et analgésiques issues d’espèces d’Hypericum natives du Rio Grande do Sul

L’objectif du présent travail était d’étudier les extraits cyclohexaniques de deux espèces d’Hypericum, natives du sud du Brésil, Hypericum caprifoliatum (HCP) et Hypericum polyanthemum (POL), qui joueraient un rôle antidépresseur et un rôle analgésique, afin de caractériser les substances responsables de ces activités, ainsi que leurs mécanismes d’action. Dans l’épreuve de la nage forcée, HCP et POL, ainsi que leurs dérivés phloroglucinols (respectivement nommés HC1 et HP4) réduisent de façon significative la durée d’immobilité des rats et des souris, indiquant un effet de type antidépresseur potentiel de ces extraits. Cet effet est totalement réversé par le SCH23390 et le sulpiride (antagonistes respectifs des récepteurs D1 et D2 de la dopamine), suggérant l’implication du système dopaminergique dans l’effet type antidépresseur de HCP et POL. D’autre part, leurs fractions riches en phloroglucinol (HC1 et HP4) inhibent la capture synaptosomale de la dopamine ([3H]-DA), de la noradrénaline ([3H]-NA) et de la sérotonine ([3H]-5HT), avec une certaine primauté d’action sur la capture de la DA. Cependant, cet effet ne semble pas dépendre d’une action directe sur les transporteurs des monoamines, puisque HC1 et HP4 n’affectent ni la liaison du [3H]-mazindol, ni celle de la [3H]-nisoxétine et ni celle du [3H]-citalopram, respectivement aux transporteurs de la DA, de la NA et de la 5-HT. Par ailleurs, nous avons évalué l’effet des extraits sur les adaptations fonctionnelles des récepteurs monoaminergiques et opioïdergiques, grâce à la liaison de [35S]-GTPγS stimulée respectivement par les monoamines et les opioïdes. Nous avons montré qu’administrés à la dose unique ou après trois traitements pendant 24 heures, HCP et POL provoquent une augmentation significative de la liaison de [35S]-GTPγS stimulée par la DA, la NA et la 5-HT, alors qu’après cinq jours de traitement, à raison de deux administrations quotidiennes, ces extraits réduisent de façon significative la liaison de [35S]-GTPγS stimulée par les monoamines. Il est important de noter que quelque soit le traitement, ni HCP, ni POL n’affecte de manière significative la liaison de [35S]-GTPγS stimulée par les opioïdes. Ces résultats démontrent que HCP et POL provoquent une altération fonctionnelle des

xxv Confidencial 23/3/2007 transmissions monoaminergiques. Nous nous sommes intéressés, par la suite, aux effets de HCP et de POL sur l’axe corticotrope, et avons montré qu’administré sur trois jours, à raison d’une administration quotidienne, HCP et POL réduisent de façon significative l’augmentation du taux de corticostérone corticale induite par le stress de la nage forcée, sans affectée de manière significative celle du taux de corticostérone sérique, ni les taux de corticostérone plasmatique et corticale basale chez les animaux non soumis aux stress. Par ailleurs, nous avons montré, qu’aux doses où ils exercent un effet type antidépresseur, HCP et POL induisent, dans le test de la plaque chaude, des effets analgésiques réversés par la naloxone (antagoniste des récepteurs opioïdes). Cet effet pourrait être attribué soit à d’autres substances, autres que HC1 et HP4, présentes dans les extraits; soit à une action indirecte de HC1 et HP4 sur les récepteurs opioïdes, puisque ni HC1, ni HP4 n’affectent de manière significative la liaison de la [3H]-naloxone. En conclusion, l’ensemble de ce travail indique que les extraits de H. caprifoliatum et H. polyanthemum développent des effets de type analgésique et antidépresseur, qui pourraient être attribués à leurs dérivés phloroglucinols. Ce dernier effet semble résulter de l’activation d’une transmission monoaminergique, selon un mécanisme d’action différent de celui des antidépresseurs de références.

Mots clés: Hypericum caprifoliatum, Hypericum polyanthemum, antidépresseur, nociception, test de la nage forcée, test de la plaque chaude, capture synaptosomale, radioliaison aux transporteurs neuronaux, [35S]-GTPγS, taux de corticosterone.

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I PARTE

INTRODUÇÃO

E

REVISÃO BIBLIOGRÁFICA

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1. INTRODUÇÃO

Os psicofármacos constituem uma classe de fármacos com fundamental necessidade de desenvolvimento. Foram introduzidos na terapêutica no final da década de 50 e propiciaram uma revolução no tratamento das doenças psiquiátricas e uma própria mudança de atitude diante destas, visto a possibilidade de uma maior compreensão de seu substrato biológico (Feighner, 1999). O conhecimento das bases neuroquímicas da depressão e esquizofrenia, por exemplo, está estritamente relacionado com o conhecimento dos mecanismos de ação de antidepressivos e antipsicóticos. No entanto, a totalidade dos fenômenos bioquímicos relacionados com estas doenças está longe de ser completamente elucidada. Da mesma forma, o mecanismo de ação exato de muitos destes fármacos ainda não é totalmente compreendido. Soma-se a isto o fato de que cerca de 35% dos pacientes psiquiátricos não respondem adequadamente ao tratamento farmacológico, e de que, mesmo a resposta terapêutica adequada, é acompanhada, na maioria das vezes, por reações adversas importantes (Graeff et al., 1999 ; Berton e Nestler, 2006).

Entretanto, a grande maioria das pesquisas é ainda alicerçada na abordagem original da teoria monoaminérgica da gênese dos distúrbios psiquiátricos, fundamentada na observação dos efeitos da reserpina, dos inibidores da monoaminoxidase e antidepressivos tricíclicos. A manutenção desta abordagem não resultará em fármacos realmente inovadores e tão pouco auxiliará na evolução do conhecimento sobre as doenças mentais (Nestler e Carlezon, 2006). Este fato torna-se preocupante considerando que distúrbios do humor, como a depressão, têm prevalência mundial de aproximadamente 18%, sendo considerada uma das doenças mentais mais incapacitantes e dispendiosas (Gold e Charney, 2002).

É neste contexto que se insere a pesquisa do gênero Hypericum. A espécie Hypericum perforatum, conhecida popularmente nos EUA e Inglaterra como St.

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John’s wort e na Alemanha como Johanniskraut (erva-de-São-João), apresenta-se como alternativa aos antidepressivos sintéticos no tratamento de depressões leves a moderadas. Estudos clínicos demonstram a eficácia de extratos padronizados de H. perforatum nestas situações (Linde et al., 1996; Bilia et al., 2002; Kasper et al., 2006) e estudos sobre seu mecanismo de ação indicam que estes extratos atuam de modo diferente dos antidepressivos atuais (Bhattacharya et al., 1998; Chatterjee et al., 1998a; Sarrell et al., 2001, Kumar et al., 2001). Uma característica interessante de sua atividade é a ação não-específica. O extrato e algumas substâncias isoladas inibem a recaptação sinaptossomal de serotonina, noradrenalina, dopamina, GABA e glutamato (Wonnemann et al., 2000; Roz e Rehavi, 2003). A ligação de histamina, de neurocinina, de corticotropina e opiódes aos seus respectivos receptores também é inibida por extratos de H. perforatum (Simmen et al., 2001).

O grupo, coordenado pelas Prof. Dr. Stela Maris Kuze Rates e Prof. Dr. Gilsane Lino von Poser, vem desenvolvendo, desde 1998, uma linha de pesquisa que visa avaliar espécies do gênero Hypericum, nativas do Rio Grande do Sul, quanto à taxonomia, constituição química e atividades biológicas, especialmente aquelas relacionadas a uma potencial atividade antidepressiva. Até o momento, foram avaliadas as espécies H. brasiliense, H. caprifoliatum, H. conatum, H. myrianthum, H. piriai, H. polyanthemum, H. carinatum e H. cordatum, sendo que H. caprifoliatum demonstrou os resultados mais promissores (Viana, 2002). Esta tese de doutorado visou aprofundar o estudo da atividade antidepressiva de H. caprifoliatum e iniciar o estudo da atividade farmacológica de H. polyanthemum, buscando identificar, em cada espécie, a(s) substância(s) ativa(s) e investigar os respectivos mecanismos da ação.

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2. OBJETIVOS

O objetivo geral deste trabalho foi estudar a ação antidepressiva e analgésica de extratos ciclo-hexano de Hypericum caprifoliatum (denominado HCP) e Hypericum polyanthemum (denominado POL) e dos derivados floroglucinol, HC1 e HP4 (uliginosina B), isolados de H. caprifoliatum e H. polyanthemum, respectivamente.

2.1. Objetivos específicos

ƒ Avaliar frações da espécie H. caprifoliatum e H. polyanthemum no modelo animal de depressão teste de natação forçada em ratos e camundongos, em diferentes tratamentos.

ƒ Estudar os possíveis mecanismos envolvidos nos efeitos observados nos modelos animais de depressão, através de: - Ensaios neuroquímicos de recaptação sinaptossomal de monoaminas; - Ligação a transportadores de monoaminas; - Avaliação do efeito sobre a funcionalidade de receptores monoaminérgicos e opióides. - Avaliação do efeito sobre os níveis séricos e corticais de corticosterona.

ƒ Avaliar o efeito antinociceptivo de extratos de H. caprifoliatum e H. polyanthemum nos testes da placa quente e contorções abdominais induzidas por ácido acético.

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3. REVISÃO DA LITERATURA

3.1. TRANSTORNOS DEPRESSIVOS

Muito do que sabemos atualmente sobre a depressão e doenças relacionadas foi descrito por médicos da Grécia e Roma antigas, que cunharam termos como melancolia e mania e perceberam sua relação. No século IV a.c, Hipócrates já fazia referência ao sofrimento e à melancolia. Ele descreveu melancolia, “a bile negra” , como um estado de “aversão à comida, falta de sono, irritabilidade, infelicidade e agitação”. Galeno (131-201 d.c.) descreveu a melancolia como “medo, depressão, descontentamento com a vida e desejo de ficar longe das pessoas”. Posteriormente, a medicina greco-romana não apenas reconheceu os sintomas da melancolia na forma de medo, desconfiança, agressão e desejos de morte, mas também a relacionou a fatores ambientais como alto consumo de vinho, “perturbações da alma por paixões”, e distúrbios no sono. Muitos textos gregos originais sobre melancolia foram transmitidos para posteridade através de textos medievais árabes, nos quais conexões entre dois principais estados de humor eram sugeridas, e supunha-se que a causa de doenças era devido às interações entre o ‘temperamento’, o ambiente e quatro ‘humores’: vento, humor, bile amarela e bile negra (WHO, 2006a).

Na era moderna, o livro “Anatomy of Melancholy” de Robert Burton (1621) foi o primeiro grande texto da história ocidental sobre as ciências cognitivas por sistematizar as descrições de muitos padrões de doenças mentais, principalmente a depressão. Burton categoriza várias formas de melancolia e tristeza, e também descreve “melancolias sem causa”. Este último significa que é possível sofrer de melancolia sem haver uma causa aparente, ou seja, evento desencadeador.

No século XIX, foram feitas várias tentativas de melhorar o conceito de melancolia e aproximá-la do que atualmente considera-se depressão. Muitos médicos, como Esquirol (1820), Samuel Tuke (1813) e Henry Maudsley (1868) (WHO, 2006a), tentaram definir a causa, natureza e forma da melancolia. No final

33 Confidencial 23/3/2007 de 1800, a ‘melancolia’ e ‘doença maníaco depressiva’ começaram a ser vistas como doenças independentes das doenças ditas ‘físicas’, de tal modo que o interesse sobre a depressão e mania foi renovado e intensos estudos sobre vários aspectos de suas características clínicas, dinâmica, neurobiologia, epidemiologia, classificação e tratamento (WHO, 2006a).

Após a II Guerra Mundial, a OMS (Organização Mundial de Saúde) criou uma força-tarefa para rever a classificação das doenças psiquiátricas e produzir uma edição revisada da Classificação Internacional de Doenças (CID). Suas revisões subseqüentes, juntamente com edições revisadas do Manual Diagnóstico e Estatístico de Transtornos Mentais (DSM) da Associação Psiquiátrica Americana, revolucionaram o modo de estudar doenças mentais. Estes guias oficiais estabeleceram critérios operacionais claros para o diagnóstico, assim como requerimentos para inclusão e exclusão, o que significa maior conhecimento sobre o que constitui a depressão e o que não a constitui (WHO, 2006a).

O Manual Diagnóstico e Estatístico de Transtornos Mentais 4ª edição com texto revisado (DSM-IV-TR, 2002), classifica os transtornos depressivos em:

- Transtorno Depressivo Maior: caracteriza-se por um ou mais episódios depressivos maiores, i.e. pelo menos duas semanas de humor deprimido ou perda de interesse, sem história de episódios maníacos ou hipomaníacos, acompanhados por quatro sintomas adicionais de depressão extraídos de uma lista que inclui: alterações no apetite ou peso, sono e atividade psicomotora; diminuição da energia; sentimentos de desvalia ou culpa; dificuldades para pensar, concentrar-se ou tomar decisões; pensamentos recorrentes sobre morte ou ideação suicida, planos ou tentativas de suicídio. O Transtorno Depressivo Maior está associado com uma alta mortalidade. Os indivíduos com Transtorno Depressivo Maior severo que morrem por suicídio chegam a 15%.

- Transtorno Distímico: sua característica essencial é um humor cronicamente deprimido que ocorre na maior parte do dia, na maioria dos dias, por pelo menos 2

34 Confidencial 23/3/2007 anos. Os indivíduos descrevem seu humor como triste ou "na fossa". Durante os períodos de humor deprimido, pelo menos dois dos seguintes sintomas adicionais estão presentes: apetite diminuído ou hiperfagia, insônia ou hipersonia, baixa energia ou fadiga, baixa auto-estima, fraca concentração ou dificuldade em tomar decisões e sentimentos de desesperança. Os indivíduos podem notar a presença proeminente de baixo interesse e de autocrítica, freqüentemente vendo a si mesmos como desinteressantes ou incapazes.

- Transtorno Depressivo Sem Outra Especificação: é incluído para a codificação de transtornos com características depressivas que não satisfazem os critérios para Transtorno Depressivo Maior, Transtorno Distímico, Transtorno de Ajustamento com Humor Deprimido ou Transtorno de Ajustamento Misto de Ansiedade e Depressão (ou sintomas depressivos acerca dos quais existem informações inadequadas ou contraditórias).

3.1.1. Dados epidemiológicos

Os Transtornos Depressivos podem acometer pessoas de qualquer gênero, raça e nível sócio-econômico. A OMS estima que atualmente 121 milhões de pessoas sofrem de depressão. Aproximadamente 5,8% dos homens e 9,5% das mulheres sofrerão um episódio depressivo em algum momento da vida, estes valores podem variar entre as diferentes populações (WHO, 2006b). A depressão crônica ou recorrente pode resultar em prejuízos de ordem pessoal e profissional. Ela é principal causa de afastamento do trabalho (medido em YLD, do inglês, Years Lived with Disability) e quarta colocada quando se considera os anos de potencial vida produtiva perdidos por morte prematura ou doença (DALY, do inglês Disability Adjusted Life Years), sendo que a OMS projeta que em 2020 a depressão alcançará o segundo lugar como causa de DALY. Além disso, o suicídio permanece uma possível decorrência do Transtorno Depressivo Maior. Os Transtornos Depressivos e a Esquizofrenia são responsáveis por 60% dos suicídios registrados no mundo (WHO, 2006b).

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Os Transtornos Depressivos estão fortemente correlacionados com fatores hereditários, considera-se que 40-50 % do risco para o desenvolvimento de depressão transmitido pelos pais, embora os genes responsáveis por este risco não estejam identificados (Berton e Nestler, 2006). A caracterização dos riscos causados pelo meio também permanece escassa, com sugestões para traumas durante a infância, estresse emocional, outras doenças que envolvam ou não o sistema nervoso central (SNC), incluindo doenças virais (Berton e Nestler, 2006).

3.1.2. Tratamentos

A primeira linha de tratamento inclui medicação antidepressiva, psicoterapia ou a combinação de ambos. Outras intervenções efetivas incluem a criação de uma rede de apoio para indivíduos, famílias ou grupos vulneráveis. As evidências sobre a prevenção de episódios depressivos são menos conclusivas. O tratamento com antidepressivos juntamente com psicoterapia é efetivo em 60 - 80 % dos pacientes. Entretanto, menos de 25 % dos indivíduos afetados (em alguns países menos de 10 %) recebem tratamento, algumas razões são a falta de recursos, de pessoal treinado e o estigma associado às doenças mentais que leva os pacientes a não procurar auxílio (WHO, 2006b).

Na década de 50 antidepressivos de duas classes foram descobertos, os tricíclicos (ADT) e os inibidores de monoaminoxidase (IMAO). Apesar de atualmente existirem mais de 25 substâncias utilizadas para fabricação de medicamentos antidepressivos, desde a descoberta dos ADT e IMAO não ocorreu nenhuma grande inovação no mecanismo de ação destes medicamentos, todos ainda agem via transmissão monoaminérgica, apenas com maior especificidade que os tricíclicos e IMAO. A grande vantagem dos antidepressivos de segunda geração e atípicos são efeitos adversos mais brandos e por isso melhor tolerados pelos pacientes. A seguir apresentamos um resumo sobre as principais características das classes de medicamentos antidepressivos disponíveis:

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3.1.2.1. Antidepressivos Tricíclicos (ADT)

No final da década de 40, Häflinger e Schindler sintetizaram uma série de mais de 40 derivados de iminodibenzila para possível uso como anti-histamínico, analgésico, anti-parkinson e/ou sedativo (Baldessarini, 1996; Nestler, 1998). Entre as substâncias selecionadas em testes pré-clínicos, por suas propriedades sedativas ou hipnóticas, estava a imipramina. Porém, durante os testes clínicos ela foi relativamente ineficaz em reduzir a agitação de pacientes psicóticos, inesperadamente, ela apresentou uma melhora indiscutível em certos pacientes deprimidos (Baldessarini, 1996). Posteriormente, descobriu-se que todos os antidepressivos tricíclicos ativos clinicamente inibem a recaptação de serotonina e noradrenalina com diferentes potências (Nestler, 1998). Porém, devido a sua origem eles também atuam sobre os receptores de histamina e acetilcolina. Esta última interação causa importantes efeitos tipo atropina, incluindo boca seca e constipação, tontura, visão borrada, sedação, hipotensão ortostática. Outras reações adversas incluem alterações cardiovasculares, em sobre-dose os ADTs diminuem a condução intraventricular podendo causar falência cardíaca ou arritmias ventriculares. Em sobre-dose estes medicamentos também podem causar ataques epiléticos principalmente em pacientes que já apresentaram episódios anteriores. Alguns exemplos são: imipramina, amitriptilina, clorimipramina, desimipramina.

3.1.2.2. Inibidores de monoaminoxidases (IMAO)

Foram os primeiros antidepressivos clinicamente ativos e tiveram um grande impacto no desenvolvimento da moderna psiquiatria biológica (Baldessarini, 1996; Stahl, 2000). Em 1951 a isoniazida e seu derivado isopropila, iproniazida, foram desenvolvidos para o tratamento de tuberculose. Observou-se que a iproniazida melhorava o humor em pacientes tuberculosos com sintomas de depressão. Em 1952, o grupo de Zeller descobriu que, diferente da isoniazida, a iproniazida possuia atividade inibidora da enzima monoamino oxidase (Baldessarini, 1996). Esta classe de antidepressivos aumenta os níveis de

37 Confidencial 23/3/2007 catecolaminas por inibir a MAO, uma das enzimas que degradam as aminas cerebrais. Os primeiros medicamentos com atividade IMAO (fenelzina, tranilcicloprida) são inibidores irreversíveis da enzima, ou seja, sua atividade retorna apenas após a síntese de uma nova enzima. Por esta razão, a ingestão de alimentos que contém tiramina, principalmente os produzidos por fermentação (p.ex. queijos, vinhos, cerveja) deve ser evitada, pois a MAO também oxida outras feniletilaminas, como a tiramina e é importante no metabolismo de primeira passagem. Quando a MAO gastrointestinal e hepática está inibida, uma grande quantidade de tiramina e outras feniletilaminas são absorvidas e alcançam a circulação sistêmica sem serem oxidadas, causando uma liberação massiva de noradrenalina e o que pode levar a hiperpirexia, taquicardia, arritimias cardíacas, crise hipertensiva severa e até mesmo hemorragia intracerebral (Hoffman e Lefkowitz, 1996; Stahl, 2000). Esta interação é menos crítica para IMAOs seletivos para MAO A e reversíveis, como moclobemida. Outras reações adversas são: hipotensão, ganho de peso e disfunção sexual. Apesar disso, alguns pacientes com depressão respondem melhor a IMAOs que a qualquer outra classe de antidepressivos (Feighner, 1999). 3.1.2.3. Inibidores seletivos da recaptação de serotonina (ISRS)

Estes antidepressivos são seletivos, mas não sub-tipo específico, aumentam a biodisponibilidade de 5-HT na fenda sináptica, tanto nos terminais como nos corpos, em todas as regiões cerebrais. Como efeito imediato os ISRS inibem o transportador de serotonina (Stahl, 1998; Yadid et al, 2000). Esta ação causa o aumento repentino de serotonina predominantemente na área somatodendrítica A administração crônica de ISRS, o aumento persistente de serotonina na área somatodendrítica do neurôrio leva a desensibilização dos auto- receptores somatodendríticos tipo 5-HT1A. Uma vez que estes auto-receptores fiquem dessensibilizados, o fluxo de impulso neural não é mais rapidamente inibido pela presença de 5-HT. Portanto, o fluxo de impulso neural é `ligado´. Outro modo de dizer-se é que, a neurotransmissão serotonérgica é desinibida, e mais serotonina é liberada do terminal axônico (Stahl, 1998). Sua eficácia, especialmente em depressão maior, não é superior aos ADT, mas o risco de

38 Confidencial 23/3/2007 sobredose é menor. Além disso, a maioria dos pacientes parece tolerar as reações adversas, que muitas vezes são transitórias como náusea, tontura, diarréia, agitação ou sedação. O uso de ISRS pode ocasionar disfunções sexuais em homens e mulheres, incluindo redução da libido, anorgasmia, retardo ejaculatório, impotência (Papakostas e Fava, 2007). Ex: fluxetina, paroxetina, sertralina.

3.1.2.4. Antidepressivos atípicos:

Inibidores seletivos da recaptação de serotonina e noradrenalina (IRSNs) - Os inibidores da recaptação de serotonina e norepinefrina são uma classe recente de antidepressivos, seu primeiro representante foi a velafaxina, mas já é um tratamento de primeira linha para depressão. Eles aumentam a atividade tanto de NA como de 5-HT, além de interagir fracamente com receptores dopaminérgicos e parecem agir como antagonista não-competitivode receptores nicotínicos (Papakostas e Fava, 2007). Parece haver uma relação dose-resposta com este fármaco, e em altas doses, o efeito adrenérgico é aumentado (Feighner, 1999). A velafaxina causa uma down-regulation aguda nos receptores β-adrenérgicos, fato que sugere um possível mecanismo de início de ação agudo, porém não existem estudos. Dependendo da dose empregada, seus efeitos adrenérgicos podem causar aumento da pressão sangüínea, entretanto este efeito não é encontrato em todos os estudos. Nos EUA o órgão de controle FDA (do inglês Food and Drug Administration) orienta que é necessária a monitorização da pressão sangüínea (Feighner, 1999). Alguns dos efeitos adversos destes antidepressivos, assim como dos ISRS, devem-se a ativação não seletiva de múltiplos receptores 5-HT e NA (Yadid et al., 2000). Outros representantes são, duloxetina e milnacipram (Papakostas e Fava, 2007).

Inibidores da recaptação de serotonina e bloqueador 5-HT2 - A este grupo pertencem a trazodona e seu análogo, a nefazodona. Elas agem como inibidores relativamente fracos da serotonina e noradrenalina (Papakostas e Fava, 2007) e bloqueiam receptores pós-sinápticos 5-HT2A/5-HT2C (Feighner, 1999; Millan, 2006). A nefazodona é mais potente e específica para serotonina que a trazodona.

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Por bloquear 5-HT2A e inibir a recaptação de serotonina, a nefazodona parece possuir um mecanismo dual de ação sobre o sistema serotonérgico. A trazodona também é um forte bloqueador do receptor adrenérgico α1, acredita-se que este receptor seja responsável pelo efeito sedativo da trazodona e também pode estar relacionado com a ocorrência de priapismo, efeito menos pronunciado durante o uso de nefazodona (Papakostas e Fava, 2007). Outras reações adversas incluem dificuldade de concentração e letargia. Ela também não possui ação do tipo quinidina, sendo segura na sobre-dose, e tem uma pequena taxa epileptigênica e de disfunção sexual, especialmente quando comparado com ISRS, venlafaxina, ADT e IMAO. A nefazodona não possui atividade anti-histamínica nem anti- colinérgica, o que melhora sua tolerabilidade e segurança. (Feighner, 1999).

Antidepressivos adrenérgico e serotonérgico específicos (NaSSa) - O exemplo de antidepressivo que atua em receptores NA e 5-HT específicos é a mirtazepina. Ela bloqueia os auto-receptores α2-adrenérgicos e α2-hetero- receptores serotoninérgicos responsáveis pela regulação na liberação de NA e 5-

HT. Além disso, bloqueia os receptores pós-sinápticos 5-HT2A , 5-HT2C e 5-HT3 (Feighner, 1999; Yadid et al, 2000). Este bloqueio resulta num aumento da atividade noradrenérgica e atividade serotoninérgica específica, o que resulta em menores efeitos colaterais do tipo ISRS (distúrbios gastrointestinais, insônia e disfunção sexual) e dos ADT (boca seca, tontura e constipação). Entretanto, a mirtazepina tem ação histaminérgica, o que pode causar sedação e aumento de apetite com aumento de peso (Feighner, 1999; Papakostas e Fava, 2007).

Inibidores da recaptação de noradrenalina (IRNs)- A reboxetina é o mais específico inibidor da recaptação de NA, com fraca afinidade pelo transportador de serotonina e dopamina e receptor muscarínico. Apesar de ser classificada como tricíclico, a desipramina também pode ser incluída nesta classe, devido a sua grande especificidade pela transportados de NA (Millan, 2006; Papakostas e Fava, 2007). O aumento de noradrenalina parece estar relacionado à ativação de receptores α1- e α2-adrenérgicos pós-sinapticos na região cortico-limbíca (Millan,

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2006). Alguns estudos sugerem que a reboxetina pode se efetiva no tratamento de alterações cognitivas e no funcionamento psicossocial durante a depressão (Yadid et al, 2000; Papakostas e Fava, 2007). A reações adversas incluem, cefaléia, insônia, boca seca, hesitação urinária e constipação. Não está associada a reações adversas típicas dos ISRS, como disfunção sexual (Yadid et al, 2000; Millan, 2006).

Inibidores da recaptação de noradrenalina e dopamina (IRND)- Seu principal representante é a bupropiona. Uma grande diferença em relação aos ISRS e IRN é a elevação dos níveis de DA no núcleo acumbens, apesar de bloquear fracamente a recaptação de dopamina (Millan, 2006). A ativação de receptores D1 que facilitam a liberação de NA podem contribuir para elevação de NA no cortex frontal (Feighner, 1999; Millan, 2006). Além disso, bupropiona é rapidamente metabolizada a derivados que inibem a recaptação de NA (Millan, 2006). Não possui efeito sobre os receptores serotoninérgicos, muscarínicos, histamínicos e α2-adrenérgicos. Porém, a bupropiona parece agir como um antagonista não-competitivo de receptores nicotínicos (Millan, 2006), este efeito pode estar relacionado com seu uso no tratamento do tabagismo (Cordioli et al., 2005). Uma vantagem em comparação aos ISRS é não estar associada com disfunções sexuais nem sedação. As reações adversas mais comuns da bupropiona são, agitação, insônia, perda de peso, boca seca, constipação, cefaléia e tremor. Um efeito adverso importante é a convulsão, principalmente em fórmulas de liberação imediata. Embora possa elevar a pressão sanguínea, este efeito não é muito comum (Papakostas e Fava, 2007).

Tianeptina - Apesar de ainda ser classificada por alguns autores como um potencializador da recaptação de serotonina (Papakostas e Fava, 2007), este efeito parece ser indireto uma vez que a afinidade da tianeptina pelo transportador de 5-HT é muito baixa (Millan, 2006) e não afeta de maneira importante os níveis extracelulares de 5-HT (Malagié et al., 2000). Ainda que o mecanismo de ação da tianeptina não esteja elucidado, vários achados experimentais justificam seu

41 Confidencial 23/3/2007 incontestável efeito antidepressivo. Cronicamente ela aumenta a sensibilidade de receptores α1-adrenérgicos, porém não modifica os níveis sinápticos de NA (Rogoz et al., 2001). O sistema dopaminérgico também é alterado pelo tratamento crônico com tianeptina, foi verificado um aumento na funcionalidade dos receptores D2 no núcleo acumbens (Dziedzicka-Wasylewska et al., 2002) e na liberação mesolímbica de dopamina (Invernizzi et al., 1992). O modo pelo qual este antidepressivo aumenta a transmissão dopaminérgica não está claro, pois ela não se liga a transportadores de DA nem aos auto-receptores D2 e D3. Outras alterações induzidas pela tianeptina que podem estar relacionadas com seu efeito antidepressivo são: atenuar a influência inibitória de GABA (ácido gama-amino butírico) e glicina sobre a excitabilidade neuronal (Kim et al., 2002); ter efeito neuroprotetor contra os efeitos do estresse causados pelos glicocorticóides e citocinas neurotóxicas e modular a transmissão glutamatérgica (McEwen et al., 2002; Castanon et al., 2004).

3.1.3. Pacientes resistentes

Um terço ou mais dos pacientes não responde e mais da metade não consegue obter ou manter uma remissão completa com qualquer tratamento farmacológico isolado. Ao avaliar a resistência de um paciente ao tratamento é preciso considerar os cinco D: diagnóstico, droga, dose, duração do tratamento e diferentes tratamentos. O médico deve considerar a mudança de todos eles caso o paciente não responda num período de 6-8 semanas. Em casos extremos, como em depressões psicóticas, catatônicas, onde o paciente corre risco de vida, podem ser empregados métodos não farmacológicos como eletroconvulsoterapia, estimulação transcraniana, privação do sono (Potter e Hollister 2006).

3.2. BASES NEUROQUÍMCAS DA DEPRESSÃO

3.2.1. Teoria monoaminérgica

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A compreensão da depressão como uma alteração na neurotransmissão cerebral está intimamente relacionada à descoberta de que fármacos podiam melhorar os sintomas de depressão. Assim, as teorias sobre a patofisiologia da depressão são derivadas de estudos sobre o mecanismo de ação de medicamentos antidepressivos e das alterações fisiológicas observadas na depressão (Henn et al., 2004). Como mencionado no item 3.1.2, os primeiros antidepressivos a serem descobertos pertenciam a duas classes de medicamentos: os tricíclicos e os inibidores de monoaminoxidases (IMAO). Os primeiros bloqueiam os transportadores de noradrenalina e serotonina, ocasionam aumento da disponibilidade dos respectivos neurotransmissores na sinapse. Os IMAO também aumentam a disponibilidade dos neurotransmissores, mas por outro mecanismo, inibindo sua metabolização pela enzima monoaminooxidase (Duman, 1999; Berton e Nestler, 2006). Por esta razão, a primeira teoria sobre a etiologia da depressão considerava que os pacientes possuíam uma carência de monoaminas, principalmente noradrenalina e serotonina. As evidências para esta hipótese eram bastante simples: certas drogas que depletavam estes neurotransmissores (p.ex. reserpina) poderiam causar depressão, enquanto os antidepressivos conhecidos na época possuíam efeitos farmacológicos de aumentar a quantidade de neurotransmissores na fenda sinaptica (Duman, 1999; Feighner, 1999; Stahl, 2000), originando a idéia de que as quantidades “normais” de monoaminas eram de algum modo depletadas causando a depressão.

Muitos estudos foram feitos para identificar na prática as deficiências teóricas de monoaminas. Ainda que essas observações sugiram que os níveis de monoaminas e a densidade de receptores e transportadores estão fortemente relacionados s com a causa e tratamento da depressão, algumas contradições sugerem um relacionamento mais complexo entre monoaminas e depressão (Maes e Meltzer, 1995; Duman, 1999). Por exemplo, os níveis de NA e 5-HT se elevam algumas horas ou dias após o início do tratamento, enquanto o efeito clínico é observado após aproximadamente 04 semanas (Duman, 1999); em indivíduos normais a depleção de 5-HT ou NA não induz sintomas de depressão

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(Miller et al., 1996). Embora a hipótese monoaminérgica seja uma noção simplificada da depressão, ela teve grande valor ao focalizar a atenção sobre os três neurotransmissores monoaminérgicos, dopamina (DA), noradrenalina (NA) e serotonina (5-HT). Isto levou a um melhor entendimento de suas funções fisiológicas e, principalmente, dos vários mecanismos pelos quais todos os antidepressivos conhecidos agem aumentando a neurotransmissão em uma ou mais destas monoaminas (Stahl, 2000).

O tratamento crônico com antidepressivos, principalmente tricíclicos, causa redução na ligação a sítios β1AR (receptores β adrenérgicos tipo 1) em regiões límbicas. Porém, várias observações mostraram que este efeito não pode ser considerado como o responsável pela ação dos antidepressivos (Duman, 1999). Primeiro, nem todos os antidepressivos possuem este efeito e o tempo para o desenvolvimento desta down-regulation é mais curto do que o para observado para o surgimento do efeito clínico. Além disso, o uso de antagonistas de βAR como anti-hipertensivos não melhora estados depressivos e pode até mesmo produzi-los em certos indivíduos, enquanto agonistas βAR produzem efeito antidepressivo em modelos animais e pacientes. O mesmo acontece com o receptor 5-HT2A: alguns antidepressivos reduzem sua expressão, em um tempo menor que o necessário para se observar efeitos clínicos, e antagonistas 5-HT2A não são antidepressivos; mas, nem todos os antidepressivos causam este efeito e o tratamento crônico com eletroconvulsoterapia causa aumento nos níveis destes receptores. Em relação aos receptores 5-HT1A, o tratamento crônico com antidepressivos causa desensibilização dos auto-receptores somatodendríticos o que leva ao aumento na transmissão serotoninérgica via receptor 5-HT1A pós- sináptico (Stahl, 1998).

Apesar dos mecanismos envolvidos na depressão e ação dos antidepressivos terem como foco principal a NA e a 5-HT, os receptores dopaminérgicos também são afetados por estes medicamentos. A maioria das evidências mostra que o tratamento crônico com antidepressivos está relacionado

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ao aumento das funções dos receptores tipo D2 (i.e. D2 e D3) e a uma diminuição no número e sensibilidade de receptores D1. Estas mudanças são mais evidentes nas áreas límbicas, i.e. aquelas áreas inervadas por neurônios dopaminérgicos da área ventral tegmental (D’Aquila et al., 2000). Considerando-se que, a depressão não pode ser explicada apenas em termos de disfunções na neurotransmissão de NA e 5-HT e que o aumento destas monoaminas na fenda sináptica por antidepressivos é apenas o primeiro passo de uma série de eventos mais complexos, nenhuma hipótese baseada em um único sistema de neurotransmissão pode ser considerada como a explicação final tanto dos efeitos terapêuticos dos tratamentos antidepressivos, como do possível substrato biológico da depressão (Duman, 1999; D’Aquila et al., 2000; Millan 2006). Existem crescentes evidências de que na depressão o sistema monoaminérgico não responde normalmente aos estímulos, sugerindo que uma deficiência na transdução do sinal, a partir da ocupação do receptor pela monoamina, pode ser a base de uma resposta celular deficiente (Stahl, 2000). Além disso, a down-regulation de receptores apóia a idéia que os receptores permanecem ativados durante o tratamento crônico com antidepressivos. Na verdade, os níveis destes receptores são reduzidos, não completamente eliminados pelo tratamento crônico com antidepressivos, sugerindo que existe um nível suficiente de receptores para responder aos níveis elevados de monoaminas. A redução no número de receptores na presença de níveis elevados de monoaminas pode ser conseqüência do aumento da funcionalidade dos receptores durante tratamentos longos. Isto pode sugerir que há uma ativação sustentada das cascatas de sinalização intracelular reguladas pelos receptores monoaminérgicos. Estas cascatas regulam muitas proteínas celulares e a expressão de genes que podem ser alvo do tratamento com antidepressivos (Duman, 1999).

3.2.2. Teoria neurotrófica

Uma das cascatas de transdução de sinal mais importantes é a que envolve as proteínas G (proteínas ligantes de guanina nucleotídeos), que são os segundos

45 Confidencial 23/3/2007 mensageiros para 80% da sinalização extracelular, incluindo hormônios, neurotransmissores e neuromoduladores (Harrison e Traynor, 2003). Os receptores associados à proteína G (GPCR) mediam respostas à estimulação de hormônios, neurotransmissores, peptídeos e aminoácidos. Quase metade das drogas conhecidas atua por meio dos GPCRs (Landry e Gies, 2003; Alberts, 2004).

As proteínas G são formadas por 03 subunidades: α, β e γ. A ligação de um agonista a um receptor associado à proteína G faz com que a subunidade α desacople GDP (5’-guanosina di-fosfato) permitindo que o GTP (5’-guanosina tri- fosfato)se ligue. A ligação do GTP causa uma mudança conformacional da subunidade α, a qual provoca a liberação do complexo βγ permite à subunidade α interagir com suas proteínas-alvo. Uma das possíveis proteínas-alvo é a adenilil ciclase, que sintetiza AMPc (3’,5’-monofosfato de adenosina cíclico) a partir do ATP (3’,5’-trifosfato de adenosina). O AMPc exerce seus efeitos na maioria das células por meio da proteína-cinase dependente de AMPc (PKA). O aumento da concentração de AMPc ativa a PKA no citosol e as subunidades catalíticas liberadas migram para o núcleo, onde fosforilam a proteína reguladora CREB (do inglês cAMP response element-binding protein). Uma vez fosforilada, a CREB estimula a transcrição gênica ao ligar-se a uma região reguladora do gene que contém uma seqüência curta de DNA chamada de CRE (do inglês: cAMP response element) (Girault e Greengard, 1999; Alberts, 2004).

Duman e col. (1997) propõem que os antidepressivos ativariam o CRE ligado ao BDNF (do inglês: brain derived neurotrofic factor) o qual teria sua expressão deficiente em pacientes deprimidos. O BDNF é o fator neurotrófico mais abundante e com maior distribuição no cérebro (Duman, 1999). Além do papel fundamental dos fatores neurotróficos na diferenciação e desenvolvimento celular, eles são necessários para sobrevivência e funcionalidade dos neurônios no sistema nervoso adulto. Diversos estudos sugerem que o BDNF pode ser um alvo de ação dos antidepressivos e que a disfunção deste e outros fatores

46 Confidencial 23/3/2007 neurotróficos pode estar relacionada à patofisiologia da depressão (Duman, 1997; Chen et al., 2001). Sob estresse, o gene para BDNF é reprimido, levando a atrofia e possível apoptose de neurônios vulneráveis no hipocampo (Sheline, 1999). A infusão de BDNF em regiões cerebrais causa efeitos similares aos de antidepressivos em modelos comportamentais como a natação forçada e o desamparo aprendido (Siuciak et al., 1997; Shirayama et al., 2002). Estes efeitos também são observados através do aumento da expressão de CREB no hipocampo (Chen et al., 2001). Além disso, a possibilidade de que os neurônios hipocampais estejam diminuídos em tamanho e prejudicados no funcionamento durante a depressão é apoiada por estudos clínicos de neuro-imagem que mostram uma redução no volume cerebral das estruturas relacionadas (Sapolsky, 2000). Isto conduziu à formulação de uma hipótese neurotrófica da depressão, consistente com um mecanismo pós ligação neurotransmissor-receptor e envolvendo uma anormalidade na expressão gênica (Duman et al., 1997; Sapolsky, 2000; Charney e Manji, 2004). Estudos demonstrando que o estresse e outras agressões do ambiente podem danificar populações específicas de neurônios e, deste modo, contribuir para patofisiologia da depressão em indivíduos vulneráveis apóiam esta hipótese (Chrousous e Gold, 1992. Duman et al., 1997; Monteggia et al., 2004; de Kloet et al., 2005, Duman e Monteggia, 2006). Por sua vez, o aumento de monoaminas na fenda sináptica induzido pelos antidepressivos irá aumentar a ativação de GPCRs levando ao aumento na concentração de AMPc e por conseqüência resultará no aumento da expressão de BDNF, que exercerá ações tróficas em neurônios alvo, aumentando assim a sobrevivência e as funções celulares. da célula (Duman et al., 1997; Duman, 1999; Duman e Monteggia, 2006).

Algumas limitações da hipótese neurotrófica da depressão incluem o fato de nem todos antidepressivos aumentarem os níveis de BDNF e que dependendo da região cerebral onde o BDNF é administrado ele pode ou não ter efeito antidepressivo. Por exemplo, a infusão de BDNF no núcleo acumbens, na área ventro-tegmental (VTA) e no sistema mesolímbico é pró-depressiva (Eisch et al.,

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2003; Duman e Monteggia, 2006). Outros receptores, fatores neurotróficos e vias de transdução de sinal também podem estar envolvidos na patofisiologia da depressão e mecanismo de ação dos antidepressivos. Um exemplo é o receptor para glutamato NMDA (N-metil D-aspartato), que influencia CREB através da ativação da proteína cinase Ca2+-dependente, sendo que o tratamento crônico com antidepressivos reduz a função deste receptor (Paul et al., 1993; Sheline et al., 1999; Bonanno et al, 2005) e a diminuição de Ca2+ intracelular normaliza a atividade neuronal na mania e na depressão (Kristhal et al., 2001). Uma vez que o BDNF e outros fatores de crescimento se ligam ao receptor tirosina cinase (TrkB) a cascata da MAPk (do inglês mitogen activated protein kinase) também é ativada (Duman, 1999; Tiraboschi et al., 2004; Tsai, 2006). Tiraboschi et al. (2004) demonstraram que o tratamento crônico com fluoxetina não influencia a fosforilação de CREB via PKA, mas que este efeito está relacionado às as cascatas de MAPk e calmodulina cinase (CaMk). Além disso, existem diferentes tipos de subunidades α nas proteínas G que podem estimular (família Gs) ou inibir

(família Gi/Gq ou Gi/Go) a produção de AMPc e, ainda, ativar outras cascatas como a do diacilglicerol (DAG), inositoltrifosfato (IP3) e fosfolipase C (PLC) (família

Gq/G11) (Duman, 1999; Harrison e Traynor, 2003).

3.2.3. Eixo hipotálamo-pituitária-adrenal

Outra hipótese importante sobre a patofisiologia da depressão é a que relaciona a desregulação do eixo hipotálamo-pituiária-adrenal (HPA) como um possível desencadeador de distúrbios do humor. A hiperatividade do eixo HPA é um dos achados mais consistentes na psiquiatria (Barden et al., 1995; Gold et al., 1995; Holsboer e Barden, 1996; Reul e Holsboer, 2002; Pariante at al 2004). Pesquisas realizadas na década de 70 mostraram uma correlação entre distúrbios do humor e níveis anormais de corticóides. Pacientes, principalmente com depressão maior, apresentam alterações como: aumento nas concentrações de cortisol no plasma e urina e de CRH (homônio de liberação da corticotropina) no fluido cérebro-espinhal (CSF); aumento no tamanho das glândulas pituitária e adrenal; aumento de resposta do cortisol ao hormônio adrenocorticotrópico

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(ACTH, do inglês adrenocorticotropic hormone) (Sachar et al., 1976; Gold et al., 1995; Nemeroff, 1996; Bremner et al., 1997). O tratamento crônico com antidepressivos leva à diminuição plasmática de cortisol (em humanos) e corticosterona (em roedores), afeta marcadores cerebrais como RNAm para CRH, aumenta a expressão de receptores para glicocortióides (GR), aumenta a função de GR e promove a translocação de GRpara o núcleo (Holsboer 2000; Pariante e Miller, 2001).

Sabe-se que o estresse psicossocial pode levar à depressão em indivíduos vulneráveis (Chrousous e Gold, 1998; Tafet e Bernardini, 2003). A adaptação ao estresse modulada por esteróides envolve a integração entre os impulsos excitatórios indicativos de estresse vindos de diferentes receptores sensórios e circuitos neurais inibitórios, que convergem para o núcleo paraventricular (PVN) do hipotálamo, influenciando o eixo HPA. Caso o controle do estresse falhe desenvolve-se um desequilíbrio entre o estímulo estressante e o retro controle negativo no nível do PVN do hipotálamo, resultando na alteração da secreção de peptídeos como o CRH, ACTH e vasopressina. Não é completamente conhecido como o desequilíbrio entre os impulsos excitatórios e inibitórios se desenvolve. O aumento na secreção de cortisol parece induzir uma diminuição de GRs hipocampais (Sapolsky, 2000). Estes receptores são considerados os mais importantes na regulação da resposta ao estresse, enquanto os receptores mineralocorticóides teriam um papel relacionado às flutuações circadianas destes hormônios (Reul e Holsboer, 2002; Pariante, 2006). A diminuição de GRs tem como objetivo contornar a quantidade excessiva de glicocorticóides, e levaria a alteração dos mecanismos de retrocontrole negativo, ou seja os níveis elevados de cortisol circulante poderiam persistir após o estímulo que deu início ao aumento de cortisol haver terminado, podendo ainda resultar em processos degenerativos no hipocampo. Desta maneira, as alterações hipocampais produzidas pela exposição prolongada a níveis excessivos de cortisol, com o conseqüente desajuste da alça de retrocontrole, podem ser responsáveis pela incapacidade dos

49 Confidencial 23/3/2007 glicocorticóides em controlar sua própria secreção durante o estresse crônico (Tafet e Bernardini, 2003).

Holsboer (2000) afirma que a teoria neurotrófica (Duman et al., 1997) e as alterações do eixo límbico-HPA são complementares e não excludentes. O aumento na ativação do receptor GR (i.e. formação de complexos cortisol-GR) levaria à dimuição na transcrição de BDNF. O GR ativado associa-se fisicamente ao CREB e assim diminui sua fosforilação. Como apenas o CREB fosoforilado (p- CREB) pode se ligar ao DNA, a redução de p-CREB resultaria na diminuição da expressão de BDNF, principalmente no hipocampo, o que diminuiria a neuroplasticidade, neurogênese e aumentaria a vulnerabilidade dos neurônios (Holsboer, 2000). Além disso, a ligação de cortisol/corticosterona aos receptores mineralocorticóides ou glicocorticóides pode regular a transcrição de genes que estão envolvidos no controle de receptores ligados à proteína G (GPCR), canais iônicos e receptores ionotrópicos (de Kloet et al., 2005).

Provavelmente a elucidação completa das causas da depressão é ainda mais complexa. Muito especialistas consideram que a depressão deve ser vista como uma síndrome e não uma doença (Gold e Charney, 2002; Tafet e Bernardini, 2003; Evans et al., 2005; Berton e Nestler, 2006; Milan, 2006). Uma das razões para isto é o fato de que diversas moléculas são partilhadas entre os sistemas nervoso central (SNC) e periférico, endócrino e imune, e estes sistemas produzirem regulação recíproca entre si (Chrousos e Gold, 1998; Tafet e Bernardini, 2003).

Assim como o hipocampo, outras estruturas límbicas também regulam o eixo HPA através da liberação de monoaminas. A adrenalina e noradrenalina pertencem ao outro importante sistema de resposta adaptativa ao estresse, o sistema autônomo simpático (SAS). Este sistema e o eixo HPA possuem uma regulação positiva entre si, i.e. a ativação de um envolve a ativação do outro (Abercrombie e Zigmond, 1995).O locus coeruleus (LC) projeta fibras

50 Confidencial 23/3/2007 noradrenérgicas para várias estruturas, incluindo a amígdala, hipocampo e PVN do hipotálamo. Juntamente com o LC, o hipotálamo lateral media a ativação do SAS (Charney et al., 1995). O sistema serotoninérgico parece exercer um controle positivo obre o eixo HPA enquanto os glicocorticóides e catecolaminas produzem alterações induzidas pelo estresse no sistema serotoninérgico (Tafet e Bernardini, 2003). As projeções serotoninérgicas a partir dos núcleos mediano (MRN) e dorsal (DRN) da rafe inervam corpos celulares produtores de CRH do PVN hipotalâmico e existem neurônios serotoninérgicos localizados inteiramente no hipotálamo (Azmitia e Whitaker-Azmitia, 1995). O sistema dopaminérgico também está relacionado à regulação em resposta ao estresse do eixo HPA (Nestler e Calerzon, 2006). Existem evidências que agonistas dopaminérgicos exerçam um controle positivo sobre o eixo HPA. A ativação de receptores dopaminérgicos cerebrais, por pergolide e análogos, leva ao aumento da concentração sérica de corticosterona em ratos e este efeito é revertido por antagonistas dopaminérgicos (Fuller et al., 1983; Foreman et al., 1989). As projeções dopaminérgicas mesolímbicas e mesocorticais estão envolvidas nos processos de adaptação. A primeira processa e reforça estimulas relacionados à recompensa e à motivação, enquando a segunda está envolvida em funções cognitivas como a avaliação de situações potencialmente estressantes (le Moal, 1995). O estresse agudo aumenta a liberação mesolímbica de dopamina (Imperato et al., 1993; Lindley et al., 1999; Dazzi et al., 2001). Experimentos de auto-estimulação no hipotálamo demonstraram sua importância nos mecanismos de recompensa (Nestler e Calerzon, 2006).

Existem fortes evidências sobre a regulação recíproca entre o sistema imune e o eixo HPA. Pesquisas mostram que a morbidade associada com depressão vai além dos distúrbios no afeto e humor; e que a mortalidade entre pacientes com depressão maior, em qualquer idade, independentemente de suicídio, uso de drogas e outros fatores prejudiciais à saúde, é duas vezes maior (Chrousos e Gold, 1998; Evans et al., 2005). Várias evidências sugeriem um mecanismo bidirecional entre as desordens do humor e outras doenças. Se aceita

51 Confidencial 23/3/2007 facilmente que doenças incapacitantes são fatores de risco para ocorrência de um episódio depressivo em pessoas vulneráveis; porém um número cada vez maior de estudos vem demonstrando que a depressão pode ser um fator de risco para doenças como cardiopatias, neoplasias, epilepsia, diabetes, dor crônica (Evans et al., 2005). Estudos mostram que pacientes deprimidos têm uma perda significativa de células do córtex frontal, uma área importante para: o discernimento entre recompensa e punição, a mudança do humor de um estado para o outro, e por exercer restrição cortical sobre a amígdala (no controle do medo) da através do eixo HPA e o SNS. Um aumento na secreção do cortisol e da noradrenalina transforma um ambiente estável em um ambiente altamente adverso em termos bioquímicos. Esta condição pode contribuir para diferentes conseqüências adversas incluindo, aumento da gordura visceral, resistência à insulina, aumento na resposta inflamatória, coagulação sanguínea aumentada, fibrólise deficiente, diminuição da formação e aumento na reabsorção óssea (Gold e Charney, 2002).

Os neurônios de CRH hipotalâmicos parecem ser os principais pontos de conexão entre eventos periféricos e respostas do SNC. Assim como a secreção de glicocorticóides é fisiologicamente aumentada em resposta a um processo inflamatório, com o objetivo de restringir o dano tecidual em conseqüência à resposta imune, dados experimentais mostram que mediadores inflamatórios/imunes ativam o eixo HPA via neurônios CRH hipotalâmicos ou via pituitária, podendo levar a respostas patológicas, como por exemplo, à depressão (Ashwell et al., 2000; Sapolsky et al., 2000). Uma hipótese para esta inter-relação seria que as citocinas e outros mediadores imunes seriam ”sensores” moleculares, responsáveis por transformar estímulos não cognitivos (i.e. processos inflamatórios) em estímulos cognitivos, permitindo que o SNC reconheça-os e elabore uma resposta integrada para os eventos periféricos (Chrousos e Gold, 1998). Ao mesmo tempo, o SNC pode reagir a tal estímulo, por exemplo, induzindo ao hipercortisolismo para inibir a resposta inflamatória (Ashwell et al., 2000). Porém, se o aumento nos níveis de glicocorticóides é patologicamente prolongado, por estresse crônico ou em indivíduos deprimidos, a imunossupressão

52 Confidencial 23/3/2007 pode levar à incidência de doenças infecciosas ou auto-imunes e a formação de tumores (Chrousos e Gold, 1998), logo, não é mera casualidade que muitas doenças auto-imunes estão associadas à depressão (Evans et al., 2005). Para facilitar a compreensão deste item por parte dos leitores franceses, um sumário, em inglês, das inter-relações entre o SNC, o eixo HPA e o sistema imune em indivíduos saudáveis e deprimidos está apresentado na figura 1.

(A)

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(B)

Figure 1: The hypothalamic-pituitary-adrenal (HPA) axis in a normal (A) and depressed (B) person. (A) The hypothalamic-pituitary-adrenal (HPA) axis is a feedback loop that includes the hypothalamus, the pituitary and the adrenal glands. The main hormones that activate the HPA axis are corticotropin-releasing factor (CRF), arginine vasopressin (AVP) and adrenocorticotropin hormone (ACTH). The loop is completed by the negative feedback of cortisol on the hypothalamus and pituitary. The simultaneous release of cortisol into the circulation has a number of effects, including elevation of blood glucose for increased metabolic demand. Cortisol also negatively affects the immune system and prevents the release of immunotransmitters. Interference from other brain regions (eg hippocampus and amygdala) can also modify the HPA axis, as can neuropeptides and neurotransmitters. (B) In depression, the hypothalamic-pituitary-adrenal (HPA) axis is upregulated with a down-regulation of its negative feedback controls. Corticotropin-releasing factor (CRF) is hypersecreted from the hypothalamus and induces the release of adrenocorticotropin hormone (ACTH) from the pituitary. ACTH interacts with receptors on adrenocortical cells and cortisol is released from the adrenal glands; adrenal hypertrophy can also occur. Release of cortisol into the circulation has a number of effects, including elevation of blood glucose. The negative feedback of cortisol to the hypothalamus, pituitary and immune system is impaired. This leads to continual activation of the HPA axis and excess cortisol release. Cortisol receptors become desensitized leading to increased activity of the

54 Confidencial 23/3/2007 pro-inflammatory immune mediators and disturbances in neurotransmitter transmission (From CNSforum Image Bank; http://www.cnsforum.com).

3.3. O GÊNERO HYPERICUM

A família Guttiferae é constituída por 50 gêneros e aproximadamente 1000 espécies distribuídas pelas regiões tropicais e subtropicais do planeta (Cronquist, 1981). Entre alguns gêneros importantes da família destacam-se Calophyllum, Garcinia, Vismia e Hypericum. Um grande número de espécies desses gêneros tem sido utilizado na medicina tradicional para o tratamento de câncer, doenças de origem viral, bacteriana e fúngica, entre outras. A ampla utilização desses vegetais tem levado à descoberta de inúmeras moléculas com diversas atividades biológicas. Entre estas, destacam-se as substâncias fenólicas. Essas substâncias - em especial as meta-diidroxiladas - freqüentemente apresentam-se substituídas por grupamentos prenila, considerados como interessantes grupos farmacofóricos. A prenilação é facilitada pela influência dos grupos hidroxila, os quais aumentam a densidade eletrônica favorecendo energeticamente as reações enzimáticas de substituição eletrofílica com pirofosfato de dimetilialila (Zuurbier et al., 1998). Posteriormente, numa seqüência biossintética, pode ocorrer ciclização da cadeia prenilada originando os derivados dimetil-benzopirânicos correspondentes.

Esse padrão de substituição – hidroxilação, prenilação e posterior ciclização do grupo prenila - é verificado nas diversas substâncias fenólicas encontradas em espécies de Guttiferae como calanolídeos, piranocumarinas com importante atividade anti-HIV-1 (Mckee et al., 1998) e xantonas, as quais têm mostrado ação antiinflamatória, anti-hepatotóxica, antiviral, antimicrobiana, antioxidante, inibidora de monoaminoxidases (IMAO), antiprotozoária e antitumoral (Rocha et al., 1994; Bennet e Lee, 1989). Outras substâncias importantes são benzofenonas, precursores de xantonas, que apresentam atividade antiprotozoária e anti-HIV-1 (Bennet e Lee 1989; Fuller et al., 1999); e derivados de floroglucinol, apresentando atividades antidepressiva, antimicrobiana, cicatrizante, antiproliferativa, entre

55 Confidencial 23/3/2007 outras (Rocha et al., 1994; Ishiguro et al., 1986; Jayasuriya et al., 1991; Rocha et al., 1996).

Em relação aos estudos farmacológicos, o gênero Hypericum, constituído por cerca de 400 espécies, tem recebido especial atenção devido à atividade antiviral de quinonas policíclicas - hipericina e pseudo-hipericina - sobre vários retrovirus, in vitro e in vivo, em particular sobre o HIV (Awang et al., 1991) e pelo emprego terapêutico de H. perforatum, a espécie mais conhecida do gênero, como antidepressivo (Linde et al., 1996; Gaster e Holroyd, 2000).

3. 3.1 Dados farmacológicos do gênero Hypericum

ATIVIDADE ANTIDEPRESSIVA

A eficácia clínica de H. perforatum foi comprovada tanto para medicamentos encontrados no comércio mundial, padronizados em hipericina (Hänsgen et al., 1994; Vorbach et al., 1994; Volz, 1997; Friede et al., 2001), como de extratos padronizados em hiperforina (Biber et al., 1998; Laakman et al., 1998) e em ambos (Philippu et al., 1999). Os medicamentos utilizados nos estudos são basicamente compostos dos seguintes extratos hidroalcóolicos; LI160: 0,2-0,3% de hipericina e 2,5-5% de hiperforina; ZE117 com 0,2% de hipericina; WS5573 e WS5572 com 0,5% e 5% de hiperforina, respectivamente; STW 0,2-0,3% de hipericina e pseudo-hipericina e 2-3% de hiperforina (Viana et al., 2001). Meta- análises (Kim et al., 1999), análise sistemática (Gaster e Holroyd, 2000) e revisões (Kasper, 2001; Käufeler et al., 2001; Bilia et al., 2002) concordam que medicamentos a base de Hypericum são mais eficazes do que placebo no tratamento de depressão leve a moderada e apresentam efeitos adversos menos pronunciados que os antidepressivos tricíclicos.

Após o estudo de Shelton e col (2001) publicado no JAMA (Journal of the American Medical Association), que não mostrou eficácia de um medicamento

56 Confidencial 23/3/2007 padronizado em hipericina no tratamento de depressão severa, houve grande controvérsia em relação ao uso de extratos padronizados de Hypericum. Um estudo brasileiro, empregando medicamento padronizado em hipericina, também demonstrou eficácia menor quando comparado ao grupo fluoxetina e placebo (Moreno et al., 2006). Contudo, estudos recentes utilizando extratos padronizados em hiperforina (WS e STW), demonstraram que estes possuem eficácia maior que placebo (Kasper et al., 2006) e comparável a inibidores seletivos da recaptação de serotonina (ISRS) no tratamento de depressão moderada a severa (Gastpar et al., 2005; Kieser et al., 2005; Szegedi et al 2005). Na revisão sistemática realizada recentemente para base de dados Cochrane, Linde e col (2005) consideram os dados em relação ao uso clínico de Hypericum são inconsistentes e confusos, principalmente para o tratamento de depressão maior, pois existe grande heterogeneidade entre os estudos placebo controlados, ainda que os estudos comparando extrato versus antidepressivos sejam mais homogêneos e mostrem eficácia equivalente.

Inicialmente, o principal constituinte a ser considerado como responsável pela atividade antidepressiva da espécie H. perforatum foi a hipericina (Suzuki et al., 1984; Wagner e Bladt, 1994; Perovic e Müller, 1995). Posteriormente, acumularam-se evidências de que a ação terapêutica depende de mais de um constituinte, entre os quais destacam-se os floruglucinóis, hiperforina e ad- hiperforina (Bhattacharya et al., 1998; Chatterjee et al., 1998a, b; Müller et al., 1998; Kaehler et al., 1999), compostos fenólicos, como procianidinas e flavonóides (quercetina, quercitrina, e hiperosídeo) (Butterweck et al., 1998, 2000; Calapai et al., 1999) e xantonas (Hölz et al., 1989; Wagner e Bladt, 1994).

O primeiro mecanismo de ação proposto para o extrato bruto foi a inibição de monoamino oxidases (MAOA e MAOB), a qual foi atribuída à hipericina (Suzuki et al., 1984). Contudo, estudos utilizando hipericina e hiperforina puras não confirmaram esta ação (Cott, 1997; Chatterjee et al., 1998a). A inibição da MAO pelo extrato bruto também não é farmacologicamente relevante, visto que não foi

57 Confidencial 23/3/2007 confirmada in vivo: Wagner e Bladt 1994) não observaram este efeito ex vivo, após a administração de 300 mg/kg de extrato de H. perforatum em ratos; estudos farmacocinéticos demonstram que os níveis sanguíneos de hipericina são de magnitude muito abaixo das concentrações necessárias para a inibição da MAO (10-3 mol/L, para hipericina e 10-4 mol/L, para o extrato bruto) (Staffeldt et al., 1994; Thiede e Walper, 1994). A inibição da catecol-o-metil-transferase (COMT) também foi demonstrada com concentrações acima dos fisiologicamente possíveis (500 µg/ml) (Thiede e Walper, 1994).

Outro possível mecanismo de ação sugerido é a inibição da recaptação de serotonina. Para extratos padronizados em hipericina foi verificada concentração inibitória 50% (CI50) de 6,2 µg/ml (Perovic e Müller, 1995). Porém, estudos recentes concluem que esta concentração seria 10 a 20 vezes maior que a dose biodisponível e que a hipericina, quando testada na concentração de 1 µM, a qual seria mais aproximada da concentração disponível in vivo, não causa inibição significativa na captação de serotonina (Raffa, 1998). Por outro lado, estudos em ratos demonstraram que o efeito do extrato alcóolico de H. perforatum é revertido ao administrar-se antagonistas serotoninérgicos (pindolol e WAY-100635) em modelo de desamparo aprendido (Gambara et al., 1999). Por outro lado, o tratamento subcrônico com extrato metanólico causa uma down-regulation de receptores β–adrenérgicos e uma up regulation de receptores serotoninérgicos em córtex frontal de ratos (Müller et al., 1997; 1998; Kientsch et al., 2001), efeito análogo ao observado após tratamento com antidepressivos tricíclicos. Nos poucos trabalhos existentes de microdiálise cerebral envolvendo extratos de H. perforatum investigam o efeito sobre os níveis cerebrais das monoaminas e seus metabólitos os resultados sobre os níveis cerebrais de DA são os mais consistentes. Diferentes tipos de extrato de H. perforatum, após administração aguda ou repetida pelas vias oral ou intraperitoneal, causam aumento nos níveis cerebrais de DA no núcleo acumbens (Di Matteo et al., 2000; Rommelspacher et al., 2001) e córtex frontal (Yoshitake et al., 2004). Apenas tratamentos repetidos foram capazes de verificar aumento nos níveis cerebrais de 5-HT no núcleo

58 Confidencial 23/3/2007 acumbens (Rommelspacher et al., 2001), enquanto no córtex frontal o tratamento agudo causa aumento significativo desta monoamina (Yoshitake et al., 2004). Nenhum trabalho encontrado observou efeito sobre os níveis cerebais de NA.

Também foi sugerido que extratos com diferentes concentrações de hipericina, hiperforina ou flavonóides, e seus respectivos derivados, atuem diferentemente nos receptores relacionados a patofisiologia da depressão (Bhattacharya et al., 1998; Butterweck et al., 1998; Calapai et al., 1999; Philippu, 2001).

A hiperforina, in vitro, inibe inespecificamente a recaptação sinaptosomal de serotonina (5-HT), dopamina (DA), noradrenalina (NA), GABA e glutamato, com

CI50 (µg/ml) de 0,11; 0,06; 0,04; 0,09 e 0,44; respectivamente (Chaterjee et al., 1998b; Kaehler et al., 1999; Müller et al., 1998, 2001). Estudos de microdiálise em ratos indicam que o tratamento com extrato padronizado em hiperforina (4,67 %) aumenta as concentrações extracelulares de dopamina, noradrenalina e serotonina (Philippu, 2001; Rommelspacher et al., 2001) e acetilcolina (Buchholzer, 2002; Kiewert et al., 2004). Este estudo de Phillipu e col. (2001) mostra ainda que extratos sem hiperforina diminuem a concentração de serotonina, enquanto que as de dopamina e noradrenalina continuam aumentadas. Estudos in vitro, empregando extratos preparados utilizando-se CO2 supercrítico (altamente lipofílicos) ricos em hiperforina (4,5 % e 38,8 %), demonstraram inibição do efeito contrátil da serotonina sobre íleo isolado de cobaio e da recaptação de serotonina por células peritoneais, indicando que alguns efeitos centrais de H. peforatum possam ser mediados por receptores 5-

HT3 e 5-HT4 (Chatterjee et al., 1998a). Outros relatos, utilizando extratos alcóolicos padronizados em hipericina, relacionaram a atividade destes extratos aos receptores dopaminérgicos, através de antagonismo farmacológico no teste de natação forçada e desamparo aprendido (Butterweck et al. 1997; Gambara et al. 1999).

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Estudos em animais demonstraram que após tratamento crônico com extrato metanólico, hipericina pura ou hipericina e procianidinas, ocorre diminuição nos níveis de corticosterona e ACTH (hormônio adrenocorticotrófico), sem alteração nos níveis de prolactina (Butterweck et al., 2001). Em um teste em voluntários, utilizando medicamento padronizado em hiperforina, foi verificado um aumento nas concentrações plasmáticas de cortisol e de hormônio do crescimento sem alteração na concentração de prolactina (Schüle et al., 2001). Em outro estudo, voluntários que receberam medicamento padronizado em hipericina e hiperforina apresentaram aumento nos níveis plasmáticos de hormônio do crescimento e diminuição nos de prolactina (Franklin et al., 2001). Esses resultados vão ao encontro de achados pré-clínicos que indicam uma ação sobre o sistema dopaminérgico, pois estas vias facilitam a liberação do hormônio de crescimento e suprimem a secreção de prolactina (Cooper et al., 1996).

Flavonóides glicosilados isolados e frações ricas em procianidinas possuem atividade no teste de natação forçada tanto no tratamento agudo quanto por 14 dias (Butterweck et al., 2000 e 2001). Calapai e col. (1999) avaliaram o efeito da administração, em ratos, de extratos contendo diferentes concentrações de flavonóides, sobre os níveis de 5-HT, NA e DA em três regiões cerebrais (córtex, diencéfalo e tronco cerebral). O extrato com maior concentração de flavonóides causou um aumento significativo nos níveis de 5-HT e NA no tronco e diencéfalo. Segundo os autores, este aumento pode indicar um modo de ação diferente para este extrato de H. perforatum, relacionado à atividade sobre eixo hipotálamo- pituitária-adrenal (HPA). Testes in vivo (Franklin et al 2000, 2004) e in vitro (Simmen et al., 2001; 2003) demonstraram que a hiperforina, a hipericina e a pseudo-hipericina possuem atividade sobre o eixo HPA, diminuindo os níveis plasmáticos de corticosterona e agindo como antagonistas do fator de liberação de corticotropina, in vitro.

Os grupos de Müller (1998, 2001) e Chatterjee (1998 e 2001) propõem que a hiperforina é o principal constituinte neuroativo de H. perforatum com potencial

60 Confidencial 23/3/2007 efeito antidepressivo e um mecanismo de ação completamente diferente dos antidepressivos atuais. Este floroglucinol age de maneira diferenciada sobre os sítios de recaptação, inibindo tanto as aminas, NA, DA e 5-HT, como os aminoácidos, L-glutamato e GABA (Chatterjeee et al., 1999). A ad-hiperforina inibe a recaptação destes neurotransmissores com potência equivalente a hiperforina (Jensen et al., 2001; Muller et al., 2001; 2003). Entretanto, estes floroglucinóis são fracos ligantes de sítios de recaptação (Gobbi et al., 2001; Jensen et al., 2001), o que conduziu à hipótese de que eles atuem em canais iônicos ligante dependente, principalmente de sódio e cálcio, e também influenciem mecanismos reguladores de pH (Singer et al., 1999; Wonnemann et al., 2000; Chatterjee et al., 2001; Marsh e Davies, 2002; Roz e Rehavi, 2003). Estes achados explicam sua não seletividade sobre os diferentes transportadores de neurotransmissores (Muller et al., 2001). Embora estes efeitos não estejam completamente caracterizados, é ainda sugerido que esta ação ocorra não nas proteínas ou outro mecanismo associado à membrana, mas sim sobre a vesícula sináptica (Kristhal et al., 2001; Roz et al., 2002) podendo a hiperforina, até mesmo, agir sobre a fluidez das membranas (Eckert e Müller; 2001).

Além da ação dos extratos sobre os receptores relacionados à teoria monoaminérgica da depressão, existem outros mecanismos que podem estar relacionados com a modulação da atividade antidepressiva de H. perforatum. Estudos em modelos animais de depressão demonstraram que ao empregarem antagonistas opióides o efeito de extratos padronizados em hipericina é antagonizado (Butterweck et al., 1997; Panocka et al., 2000). Extratos brutos de H. perforatum reduzem a expressão de citocinas (Thiele e Walper et al., 1994; Calapai et al., 2001), que têm sido consideradas como relevantes na etiologia da depressão (Nestler, 1998).

Existem poucos relatos sobre a avaliação de outras espécies com possível ação antidepressiva. H. calycinum, H. canariense, H. glandulosum, H.

61 Confidencial 23/3/2007 grandifolium e H. reflexum foram ativos no modelo animal de depressão Teste de Natação Forçada (Özturk et al., 1996; Sanchez-Mateo et al. 2002). Entre as espécies brasileiras apenas H. brasiliense havia sido estudado, apresentando atividade inibidora de monoamino oxidase in vitro (Rocha et al., 1994).

AÇÃO ANALGÉSICA

Experimentos utilizando extratos alcoólicos de H. perforatum demonstraram atividade analgésica no teste de tail-flick (Özturk et al., 1996, Kumar et al., 2001), placa aquecida e teste das contorções abdominais induzidas por ácido acético (Kumar et al., 2001). Entretanto, outro estudo utilizando o teste de tail-flick e o da placa quente apresentou resultados negativos para o extrato hidro-alcóolico de H. perforatum (Gambara et al., 1999). Experimentos in vitro indicaram um possível efeito analgésico para hiperforina, uma vez que ela bloqueia canais de sódio e inibe receptores do tipo ATP dependentes, envolvidos na transdução do estímulo de dor (Kristhal et al., 2001). Estudos de binding demonstraram afinidade de hipericina (1µM) por receptores opióides tipo σ, com inibição de binding específico de 48 % (Raffa, 1998). Em um estudo com medicamento padronizado em hipericina sobre o efeito em dor neuropática em humanos não houve diferença em relação ao placebo (Sindrup et al., 2000).

Outras espécies que demonstraram atividade antinociceptiva em modelos animais, tail-flick, teste da formalina e teste das contorções abdominais induzidas por ácido acético foram H. calycinum e H. triquefolium (Özturk et al., 1996; Apaydin et al., 1999; Trovato et al., 2001). As espécies H. canariense H. glandulosum H. reflexum além do efeito analgésico também apresentaran atividade antiinflamatória (Rabanal et al., 2005; Sanchez-Mateo et al., 2006). H. brasiliense apresentou atividade no teste da placa aquecida e tail-flick (Mendes et al., 2002).

AÇÃO ANSIOLÍTICA

62 Confidencial 23/3/2007

Estudos de binding, com um extrato alcóolico de H. perforatum, demonstraram afinidade por receptores de adenosina, benzodiazepínicos, GABAA e GABAB , com Ki (µg/ml) de 1; 24; 0,075 e 0,006 respectivamente (Cott, 1997). A atividade ansiolítica in vivo, foi verificada em extratos de H. perforatum padronizados tanto em hiperforina quanto em hipericina (Bhattacharya et. al., 1998; Chatterjee et al., 1998a; Vandenbogaerde et al., 2000; Coleta et al., 2001). A hiperforina em baixas doses (1mg/kg) e seu derivado sintético, acetato de hiperforina (3-5 mg/kg) possuem efeito ansiolítico no teste do labirinto em cruz (Chatterjee et al., 1998a; Zanoli et al., 2002) enquanto o extrato lipofílico apresenta este efeito apenas em doses altas (300mg/kg) (Bhattacharya et al., 1998). Uma vez que o aumento nos níveis extracelulares de GABA causado por hiperforina in vitro (Chatterjee et al., 1998a) não foi observado in vivo (Kaehler et al., 1999), Chatterjee e col. (1998a) argumentam que o efeito ansiolítico apresentado pela hiperforina possa ser causado pela ação sobre receptores serotoninérgicos tipo 5-HT3 e 5-HT4, relacionados à atividade ansiolítica (Barnes e Sharp, 1999). Esta inferência deve-se ao fato de que, quando testada em preparação de íleo isolado de cobaio, a hiperforina inibiu a contração do órgão induzida por serotonina (Chatterjee et al., 1998a). No íleo são encontrados receptores 5-HT3 e 5-HT4, relacionados à atividade ansiolítica (Barnes e Sharp, 1999).

Vandenbogaerde e col. (2000), utilizando técnicas de patch-clamp, relacionaram a ação ansiolítica à atividade da hipericina e pseudo-hipericina, demonstrando que esta estimula receptores GABAA e inibe a transmissão glutamatérgica mediada por receptores NMDA. O extrato aquoso de H. perforatum também apresentou atividade ansiolítica, a qual os autores relacionaram a presença de flavonóides e hipericina, uma vez que o extrato não possuía hiperforina (Coleta et al., 2001).

Testes utilizando modelos comportamentais diferenciados, como o labirinto em T, The Mouse Defense Test Battery (MDTB) e o teste de esconder esferas

63 Confidencial 23/3/2007

(marble-burying test), mostraram que o extrato LI 160 possui potencial efeito no tratamento de síndrome do pânico e transtorno obsessivo compulsivo (Flausino et al., 2002; Beijamini e Andreatini, 2003; Skalisz et al., 2004).

AÇÃO ANTIMICROBIANA E ANTIPARASITÁRIA

Em 1954, Neuwald e Hagenström (apud Reichling et al., 2001) reportaram atividade contra Staphylococcus aureus para os extratos éter de petróleo e acetona de H. perforatum. Na Rússia, extratos acetônicos são usados clinicamente para tratar infecções bacterianas e destes extratos, foi isolada e identificada a hiperforina (Bystrov et al., 1975 apud Reichling et al., 2001), a qual foi ativa contra bactérias gram-positivas, mas inativa contra gram-negativas e fungos (Gurevich et al., 1971 apud Reichling et al., 2001; Ishiguro et al., 1986; Jayasuriya et al., 1991; Tada et al., 1991; Yamaki et al., 1994; Rocha et al., 1995). Floroglucinóis extraídos de H. papuanum foram ativos contra Bacillus cereus, Staphylococcus epidermidis e Micrococcus luteus (Winkelmann et al., 2000). A atividade antimicrobiana de H. perforatum também está relacionada com a presença de óleos voláteis e flavonóides (Maisenbacher e Kovar, 1992; Trifunovic et al., 1998). Reichling e col. (2001) encontraram atividade contra bactérias gram- positivas, até mesmo para Staphylococcus aureus resistente a meticilina, em infusões preparadas com as partes aéreas de H. perforatum.

Saroaspidina A, B, C e sarotroleno C e D, floroglucinóis de H. japonicum, apresentaram efeito antibiótico in vitro (Ishiguro et al., 1987; Ishiguro et al., 1994). Em H. calycinum, foi identificado um floroglucinol com atividade antimalárica e antifúngica (Decosterd et al., 1991). Um extrato alcóolico de H. brasiliense demostrou atividade inibitória sobre bactéria B. subtillis em cromatografia bioautográfica (Rocha et al., 1995; Rocha et al., 1996).

As xantonas identificadas em H. roeperanum possuem atividade antifúngica contra Candida albicans (Rath et al., 1996), e as xantonas presentes em H. patulum demonstraram atividade antimicrobiana (Ishiguro et al., 1996). Extrato

64 Confidencial 23/3/2007 metanólico de H. patulum também apresentou eficácia comparável ao nitrofural em modelo de cicatrização em ratos (Mukherjee et al., 2000) e floroglucinóis isolados desta espécie apresentaram atividade antimicrobiana (Winkelmann et al., 2001). Os óleos voláteis de H. scabrum, H. scabroides, H. triquetrifolium (Kizil et al, 2004), H. rumeliacum (Couladis et al., 2003), H. hyssopifolium e H. lysimachioides (Toker et al., 2006) também possuem atividade antibacteriana in vitro.

AÇÃO ANTIVIRAL

Estudos mostraram que hipericina e pseudo-hipericina inibem uma variedade de vírus encapsulados, inclusive herpes simples dos tipos 1 e 2, vírus de imunodeficiência humana tipo 1 (HIV-1), vírus para-influenza 3 e citomegalovírus (Wood et al., 1990; Taylor et al., 1996; Axarlis et al., 1998). Estudos clínicos com hipericina em pacientes com SIDA (síndrome da imunodeficiência adquirida) demonstraram redução na carga viral, sem sinais de toxicidade nem fotosensibilidade cutânea. Infusões de H. perforatum, contendo flavonóides, saponinas, ácidos fenólicos, taninos e polissacarídeos(Serkedjieva et al., 1990), e extratos de H. mysorense, H. hookerianum (Vijayan et al., 2004) apresentaram atividade contra vírus de herpes simples tipo 1 e influenza tipo A e

B.

ATIVIDADE CITOTÓXICA

A hipericina é a principal substância fototóxica de H. perforatum (Wilhelm et al., 2001). Quando foto-ativada, a hipericina é citotóxica in vitro e in vivo, sendo um potencial agente fotossensibilizador para o tratamento de tumores na terapia fotodinâmica (Vandenbogaerde et al., 1996; Ali et al., 2001). Existem relatos de que a hipericina inibe o crescimento de linhagens de células de glioma através da inibição da proteína cinase C (Couldwell et al., 1997). Ensaios in vitro indicam que a hipericina é um potente antagonista da ligação de topoisomerase II e/ou da clivagem de DNA (Peebles et al., 2001). Porém também existem certas linhagens de células tumorais contra as quais o extrato foi ativo, mas hipericina e hiperforina

65 Confidencial 23/3/2007 não, como a K562 (células eritroleucêmicas) (Roscetti et al., 2004), T24 e NBT-II (câncer de bexiga) (Skalkos et al., 2005). Outras espécies que apresentaram efeito citotóxico foram H. papuanum e H. sampsonii (Hu et al., 1998; Winkelmann et al., 2001).

ATIVIDADE SOBRE O SISTEMA ENZIMÁTICO CITOCROMO P450 E GLICO PROTEÍNA P

Em 1999 Ernst questionou a segurança do uso medicamentos a base de Hypericum perforatum devido a vários relatos de caso mostrando interações entre estes e medicamentos, principalmente contraceptivos orais, imunossupressores, anticoagulantes anti-histamínicos e digitálicos. Estudos clínicos verificaram que pacientes utilizando H. perforatum apresentam diminuição nos níveis plasmáticos de medicamentos metabolizados pelo sistema enzimático citocromo P450 (CYP450), indicando que os extratos causam uma indução deste sistema (Mai et al., 2004; Herbert et al., 2004). Estudos in vitro demonstraram que esta indução poderia ser causada pela ativação do receptor nuclear pregnano X, o qual induz a expressão da sub-enzima CYP3A4 (Moore et al., 2000). Contudo, as conseqüências da ação de H. perforatum sobre o CYP450 ainda são controversas (Jobst et al., 2000; de Smet e Touw, 2000; Wheatley, 2000; Yue et al., 2000). Obach (2000) verificou que o extrato bruto, hiperforina, hipericina e I3-II8- biapigenina inibem competitiva e não-competitivamente a atividade de diferentes enzimas do CYP450. Existem estudos em animais que verificaram ativação das enzimas do sistema CYP450 (Durr et al., 2000; Krusekopf et al., 2003) enquanto outro obteve resultado negativo (Nölder & Chatterjee, 2001). Um estudo em humanos, demonstrou que a co-administração de medicamento a base de H. perforatum não alterou o perfil farmacocinético de dextrometofano (metabolizado pelo CYP2D6) nem de alprazolam ou midazolam (metabolizados pelo CYP3A4) (Markowitz et al., 2000; Hall et al., 2003). Entretanto, no estudo de Hall e col. (2003) assim como em outros (Pfrunder et al., 2003, Murphy et al., 2005) a co- administração de H. perforatum causou a redução da área sobre a curva, da meia- vida de contraceptivos orais e sangramentos no meio do ciclo. Além do efeito

66 Confidencial 23/3/2007 sobre CYP3A4, não pode ser descartada a possibilidade de inibição das outras isoenzimas do complexo CYP450 e da influência em processo de fase II do metabolismo (sulfatação, acetilação ou glicuronidação) (Marowitz et al., 2000). Outro evento que pode determinar a redução da biodisponibilidade de alguns medicamentos co-administrados com H. perforatum é sua atividade indutora da glicoproteína P, ocasionando a redução na absorção intestinal, como é o caso da digoxina (Durr et al., 2000; Wang et al., 2004; Tian et al., 2005).

Revisões recentes confirmam que extratos de H. perforatum e alguns de seus principais metabólitos, hipericina e hiperforina, induze a glicoproteína P intestinal e a enzima CYP 450 hepática e intestinal, reduzindo a concentração plasmática de alguns medicamentos como, agentes antineoplásicos, anti- coagulantes, anti-hitamínicos, contraceptivos orais, digitálicos, imunossupressores, (Zhou et al., 2004; Mannel, 2005; Singh, 2005). Apesar disto, os extratos são seguros quando prescritos por médicos que conheçam suas possíveis interações medicamentosas (Knuppel e Linde, 2004; Mannel, 2005).

OUTRAS ATIVIDADES

Extratos de H. perforatum tem efeito nootrópico (Kumar et al., 2000; Khalifa 2001). Klusa e col (2001) demonstraram que baixas doses de extrato etanólico (50 mg/kg, v.o.) de H. perforatum facilitam a consolidação de memória em animais e que a hiperforina pura (1,25 mg/kg, v.o.) apresenta maior potência como agente nootrópico do que como antidepressivo no modelo de esquiva passiva (Klusa et al., 2001). Em experimentos in vitro, a hiperforina em concentrações nanomolares parece apresentar efeito neuroprotetor por inibir canais voltagem-dependente de Ca++, NMDA, AMPA e purinérgicos, que são alvos farmacologicamente importantes para prevenir a excitotoxicidade causada pela saturação de Ca++. Enquanto que em altas concentrações (micromolar), este metabólito secundário aumenta o cálcio intracelular (Chatterjee et al., 1999; Krishtal et al., 2001). Um estudo piloto, em pacientes sofrendo de fatiga, demonstrou redução dos sintomas após 6 semanas de tratamento com medicamento a base de extrato metanólico de

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H. perforatum padronizado em hipericina. Juntamente com as medidas de fadiga foi empregada uma escala de ansiedade e depressão (Hospital anxiety and depression scale) a qual foi significativamente reduzida durante este período (Stevinson et al., 1998).

Neary e col. (2001) demonstraram que o extrato LI 160 (5 % de hiperforina) ativa uma das proteínas da cascata da MAPkinase (mitogen induced protein kinase), envolvida na regulação gênica relacionada à proliferação e sobrevivência celular (Kolch, 2000).

Extratos hidroalcóolicos de H. perforatum reduzem o consumo de álcool em linhagens de ratos geneticamente selecionadas para preferirem etanol (10 %) a água. Nestas linhagens, que normalmente possuem alta tendência à depressão, o tratamento com os extratos diminuiu o tempo de imobilidade no teste de natação forçada, juntamente com a redução de consumo de álcool, podendo, portanto, seu efeito sobre o consumo de etanol estar relacionado com sua ação antidepressiva (De Vry et al.,1999; Carai et al., 2000; Pandocka et al., 2000; Perfumi et al., 2005).

Floroglucinóis de H. chinense e H. erectum demontraram atividade inibitória de leucotrieno D4 e tromboxano A2, o que pode indicar uma atividade antialérgica (Tada et al., 1991). Frações de H. perforatum ricas em procianidinas antagonizaram o efeito de vasocontração induzido por histamina e prostaglandina

F2α em artérias, podendo este efeito estar relacionado a inibição de fostodiesterases e/ou da enzima conversora de angiotensina (Melzer et al., 1991).

3.4. REVISÃO DOS DADOS DE ESPÉCIES DE HYPERICUM NATIVAS DO RIO GRANDE DO SUL

3.4.1 Dados químicos

Os estudos fitoquímicos, realizados no laboratório de Farmacognosia da Faculdade de Farmácia - UFRGS, verificaram a pesença das seguintes substâncias:

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Derivados de floroglucinol: Até o momento, todos os derivados de floroglucinol identificados apresentam estrutura dimérica consistindo de um ácido filicínico ligado a um floroglucinol. A partir do extrato ciclo-hexano das partes aéreas de H. myrianthum, foram isolados os floroglucinóis já descritos: japonicina A (Dall’ Agnol et al., 2003), presente em H. japonicum (Ishiguro et al., 1987) e H. brasiliense (Rocha et al., 1995), e uliginosina B (Ferraz et al., 2002a) também presente em H. carinatum e H. polyanthemum (Nör et al., 2004). Hiperbrasilol B, previamente isolado de H. brasiliense (Rocha et al., 1996), foi identificado em H. carinatum e H. caprifoliatum. A partir de H. caprifoliatum foi também isolado um conjunto de tautômeros, cuja análise espectroscópica por ressonância magnética nuclear (RMN) demonstrou tratar-se de um derivado de floroclucinol ligado ao ácido filicínico (Viana, 2002). A separação e elucidação estrutural destes tautômeros estão ainda em andamento.

Benzopiranos: A partir do extrato clorofórmico das partes aéreas de H. polyanthemum, três benzopiranos de estrutura inédita foram isolados: HP1 (6- isobutiril-5,7-dimetóxi-2,2-dimetil-benzopirano), HP2 (7-hidróxi-6-isobutiril-5- metóxi-2,2-dimetil-benzopirano) e HP3 (5-hidróxi-6-isobutiril-7-metóxi-2,2-dimetil- benzopirano) (Ferraz et al., 2001). Posteriormente, estes benzopiranos também foram isolados de H. ternum (Ferraz et al., 2005c). Utilizando a técnica de micropropagação in vitro, Bernardi e col. (2005a) conseguiram que as plântulas de H. polyanthemum produzissem benzopiranos. Estas plântulas foram aclimatizadas com sucesso e mostraram o mesmo perfil de benzopiranos encontrado na planta selvagem (Bernardi et al. 2005a).

Taninos e flavonóides: A avaliação de taninos demonstrou teores entre 5 a 16%; H. myrianthum (5,1%), H. caprifoliatum (6,4%), H. polyantemum (6,7%), H. carinatum (9,1%), H. connatum (11,5%), H. ternum (16,7%) (Dall’ Agnol et al., 2003). A fração fenólica não-tanante destas espécies foi analisada quanto à presença de flavonóides glicosilados. Foram encontrados os compostos

69 Confidencial 23/3/2007 normalmente citados na literatura: hiperosídeo, quercitrina, isoquercitrina, guaijaverina (Dall’ Agnol et al., 2003).

Benzofenonas: A partir das partes aéreas de H. carinatum, foram isoladas duas benzofenonas inéditas, carifenona A e carifenona B (Bernardi et al., 2005b).

Óleos voláteis: As espécies de Hypericum não são caracteristicamente aromáticas, nas espécies H. caprifoliatum, H. polyanthemum, H. myrianthum, H. carinatum, H. connatum and H. ternum a quantidade de óleos voláteis varia de 0,1% a 0,5%. Nestas espécies, sesquiterpenos estão em maior concentração que monoterpenos e todas possuem alcanos, principalmente nonano e undecano (Ferraz et al., 2005a).

Após investigação fitoquímica de oito espécies, H. brasiliense, H. carinatum, H. caprifoliatum, H. connatum, H. cordatum, H. myrianthum, H. polyanthemum e H. piriai, Ferraz et al. (2002b) verificaram a ausência de hipericina em todas as espécies. Este resultado está de acordo com a divisão quimiotaxonômica proposta por Robson (1990), onde a produção de hipericina é relacionada com a presença de glândulas negras nas folhas. As espécies brasileiras, que pertencem às seções Brathys e Trigynobrathys possuem apenas glândulas pálidas, que não produzem hipericina.

3.4.2 Dados biológicos

ATIVIDADE ANTIPROLIFERATIVA E ANTIOXIDANTE

Ferraz e colaboradores (2005 b, c), analisaram o efeito antiproliferativo dos extratos brutos e frações (hexano, clorofórmio e metanol) de seis espécies de Hypericum nativas do sul do Brasil (H. caprifoliatum, H. carinatum, H. connatum, H. myrianthum, H. polyanthemum e H. ternum) e de três benzopiranos (HP1, HP2 e HP3) de H. polyanthemum. Extratos e frações de H. caprifoliatum, H. myrianthum, H. ternum e os benzopiranos apresentaram atividade antiproliferativa

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contra as células de carcinoma de cólon humano HT-29, células não pequenas de carcinoma de pulmão H-460 e células de glioma humano maligno U-373.

As benzofenonas carifenona A e B, isoladas de H. carinatum, foram testadas no teste de TRAP (do inglês: total radical-trapping parameter assay), apenas carifenona A apresentou atividade antioxidante (Bernardi et al., 2005b).

ATIVIDADE ANTIMICROBIANA E ANTIVIRAL

Extratos metanólicos totais de H. caprifoliatum, H. carinatum, H. connatum, H. myrianthum, H. polyanthemum , H. ternum, benzopiranos e derivados de floroglucinol, nas concentrações de 100 e 200 µg/ml, foram testados, através do método de difusão em agar, contra as seguintes bactétias: Escherichia coli, Bacillus cereus, B. subtilis, Micrococcus luteus, Staphylococcus aureus, S. epidermidis; e os fungos Candida albicans e Saccharomyces cerevisiae (Dall’Agnol et al.,2003; 2005). Os extratos que apresentaram maiores halos de inibição contra S. aureus foram H. caprifoliatum e H. myrianthum, em ambas as concentrações. H. polyanthemum (100 e 200 µg/ml) e H. ternum (200 µg/ml) foram ativos contra B.subtilis e M. luteus

Fenner e col. (2005) utilizaram o método de diluição em ágar para testar extratos de seis espécies (H. caprifoliatum, H. carinatum, H. connatum, H. myrianthum, H. piriai, H. polyanthemum e H. ternum) contra fungos dos gêneros Candida, Saccharomyces, Cryptococcus, Aspergillus, Epidermophyton, Trichophyton e Microsporum. O extrato clorofórmico de H. ternum apresentou maior atividade antifúngica principalmente contra espécies do gênero Candida

(MICs varando de 250 a 1000 µg/ml) e T. mentagrophytes e T. rubrum (MIC50=250 µg/ml).

O extrato metanólico de H. connatum apresentou atividade inibitória in vitro sobre o crescimento de vírus da imunodeficiência felina (Schmitt, 2000; Schmitt et al., 2001).

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ATIVIDADE ANALGÉSICA

O efeito antinociceptivo dos extratos ciclo-hexânico e metanólico de H. caprifoliatum (90 mg/kg) foi avaliado nos testes da placa aquecida (i.p. e v.o) e no teste de contorções abdominais induzidas por ácido acético 0,8%. Ambos os extratos apresentaram atividade no teste da placa aquecida. O efeito dos extratos ciclo-hexânicos de ambas as espécies parece estar relacionado com o sistema opióide, pois o efeito no teste da placa aquecida foi completamente bloqueado com o pré-tratamento com naloxona (antagonista opióide). Por outro lado, o efeito analgésico do extrato metanólico é apenas parcialmente relacionado a este sistema, uma vez que o bloqueio causado por naloxona foi apenas parcial para via i.p. e que o efeito analgésico v.o. não foi afetado. No teste de contorções abdominais induzidas por ácido acético 0,8% apenas o extrato ciclo-hexânico apresentou atividade (Viana, 2002). Estes resultados juntamente com os obtidos nesta tese encontram-se encartados no Capítulo 1 (Viana et al., 2003).

ATIVIDADE ANSIOLÍTICA

O extrato ciclo-hexânico de H. caprifoliatum (ECH 90 mg/kg v.o.) foi testado em três modelos animais: labirinto em cruz elevada, potenciação do sono barbitúrico (pentobarbital 40 mg/kg, i.p.) e proteção a convulsões induzidas por pentilenotetrazol (PTZ, 80 mg/kg, i.p.). ECH não alterou significativamente os parâmetros observados no labirinto em cruz elevada, apesar de o tempo de permanência e o número de entradas no braço aberto terem sido maiores que para o controle negativo. O extrato prolongou significativamente a duração do sono, mas não alterou o tempo de indução. ECH não protegeu contra as convulsões induzidas por PTZ (Viana, 2002). Paralelamente foram realizados ensaios com íleo isolado de cobaio com o extrato ciclo-hexânico sem cera (HCP). O extrato (10 µg/ml) demonstrou ser um antagonista não-competitivo de receptores serotoninérgicos periféricos (5-HT3 e 5-HT4). Este resultado pode indicar que ECH possui um efeito ansiolítico, porém com mecanismo de ação diferente dos benzodiazepínicos (Viana, 2002), uma vez que o bloqueio de

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receptores 5-HT3 está relacionado ao efeito ansiolítico de algumas substâncias (Graeff, 1999). Estes resultados encontram-se no Capítulo 1 (Viana et al., 2006).

ATIVIDADE ANTIDEPRESSIVA

As espécies H. caprifoliatum, H. carinatum, H. connatum, H. myrianthum, H. piriai, H. polyanthemum e H. cordatum, e três benzopiranos isolados de H. polyanthemum (HP1; HP2; e HP3) foram testadas quanto à atividade inibidora de monoamino oxidase A e B em preparações de mitocôndrias de cérebro de ratos.

Os extratos que apresentaram inibição significativa apenas para MAOA, os com maior atividade na concentração de 1,5x10-2 mg/ml foram o extrato clorofórmico de H. caprifoliatum (83%)e de H. polyanthemum (82%) e o extrato éter de petróleo de

H. piriai (90%), entre os benzopiranos apenas HP3 (IC50 MAOAde 22,2µM) apresentou atividade significativa (Gnerre et al. 2001). As observações in vitro não foram confirmdas in vivo, a única espécie que apresentou redução no tempo de imobilidade dos ratos foi H. caprifoliatum, mas não o extrato clorofórmico, e sim o éter de petróleo (Daudt et al., 2000; Gnerre et al. 2001).

O possível mecanismo de ação da espécie H. caprifoliatum está sendo investigado e, até o momento, os dados indicam o envolvimento do sistema dopaminérgico. Verificou-se que o efeito antiimobilidade do extrato lipofílico (270 mg/kg/dia, v.o.) é prevenido pelo pré-tratamento com sulpirida (50 mg/kg, i.p.) (antagonista D2) e que este extrato (90 mg/kg, v.o.) potencializa a hipotermia causada por apomorfina (16 mg/kg, s.c.) (Viana, 2002). Estes resultados juntamente com os obtidos nesta tese encontram-se encartados no Capítulo 1 (Viana et al., 2005).

Para facilitar a compreensão deste item por parte dos leitores franceses, um resumo, em inglês, das atividades farmacológicas das espécies de Hypericum nativas do Rio Grande do Sul e de H. perforatum estão apresentadas na tabela 1.

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Table 1: Pharmacological activities of Hypericum species.

HYPERICUM PHARMACOLOGICAL ACTIVITY SPECIES H. brasiliense . EtOH: antibacterial (Rocha et al., 1995; Rocha et al., 1996) and antinociceptive (Mendes et al., 2002) HCP: antinociceptive (Viana et al., 2003), antidepressant (Viana et al., 2005) and antibacterial (Dall’Agnol et al., 2003, 2005) H. caprifoliatum HC1: antidepressant (Viana et al., 2005) MET: antinociceptive (Viana et al., 2003), antiproliferative (Ferraz et al., 2005 b) and antibacterial (Dall’Agnol et al., 2003) H. carinatum benzophenone Carifenone A: antioxidative (Bernardi et al., 2005b) H. connatum MET: antiviral (Schmitt et al., 2001) H. myrianthum MET: antiproliferative (Ferraz et al., 2005 b) and antibacterial (Dall’Agnol et al., 2003) POL and HP4: antidepressant (Chapter 3) and antinociceptive H. polyanthemum (Viana et al., 2003) HP2, HP3: antiproliferative (Ferraz et al., 2005 c) and antibacterial (Dall’Agnol et al., 2005) H. ternum MET: antiproliferative (Ferraz et al., 2005 b), antibacterial (Dall’Agnol et al., 2003) and antifungal (Fenner et al., 2005) extracts, essential oils, xanthones hyperforin: antimicrobial (for review see Reichling et al., 2001) LI 160, hyperforin: antidepressant (Linde et al., 1996; Kasper et al., 2006), anxiolytic (Chatterjee et al., 1998a; Beijamini e Andreatini, 2003; Skalisz et al., 2004), CYP450 and Pgp inducer (Herbert et al., 2004; Tian et al., 2005) H. perforatum EtOH: antinociceptive(Özturk et al., 1996, Kumar et al., 2001), nootropic (Klusa et al., 2001) hypericin: antidepressant (Linde et al., 1996), anxiolytic (Chatterjee et al., 1998a; Beijamini e Andreatini, 2003; Skalisz et al., 2004), CYP450 and Pgp inducer (Herbert et al., 2004; Tian et al., 2005), citotoxic (Vandenbogaerde et al., 1996; Ali et al., 2001) and antiviral (Axarlis et al., 1998). EtOH: ethanolic extract HCP: H. caprifoliatum cyclohexane extract HC1: H. caprifoliatum phloroglucinol derivative MET: methanolic extracts POL: H. polyanthemum cyclohexane extract HP4: H. polyanthemum phloroglucinol derivative LI 160: H. perforatum standardized extract CYP450: cytochrome 450 Pgp: glycoprotein P

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II PARTE

RESULTADOS EXPERIMENTAIS

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1.1.

CAPÍTULO 1: Artigos publicados

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1.1. INTRODUCTION

In this chapter three papers are enclosed. The first one that has been published in 2003 in Brazilian Journal of Medical and Biological Research concerns the antinociceptive effects of Hypericum caprifoliatum and Hypericum polyanthemum extracts. The second paper, published in 2005 in Neuropharmacology, reports the activity and mechanism of action of Hypericum caprifoliatum cyclohexane extract as antidepressant agent. The last paper (Fundamental and Clinical Pharmacology) reviews chemical and pharmacological published results as well as original data describing the effects of Hypericum caprifoliatum extracts on depression, anxiety and analgesia.

79 Brazilian Journal of Medical and Biological Research (2003) 36: 631-634 Antinociceptive activity of Hypericum spp 631 ISSN 0100-879X Short Communication

Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae)

A.F. Viana1,2, 1Laboratório de Psicofarmacologia, Departamento de Produção de Matéria Prima, A.P. Heckler1, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, R. Fenner1 and RS, Brasil S.M.K. Rates1,2 2Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil

Abstract

Correspondence The aim of the present study was to assess the analgesic activity of the Key words S.M.K. Rates aerial parts of two Hypericum species native to Southern Brazil, H. · Hypericum caprifoliatum Faculdade de Farmácia, UFRGS caprifoliatum and H. polyanthemum. The antinociceptive effect of the · Hypericum polyanthemum Av. Ipiranga, 2752 H. polyanthemum cyclohexane extract (POL; 180 mg/kg) and of the H. · Analgesia 90610-000 Porto Alegre, RS caprifoliatum methanol (MET) and cyclohexane (CH) extracts (90 · Antinociception Brasil mg/kg) was evaluated in the hot-plate (ip and po) and writhing (po) · Hot-plate test Fax: +55-51-3316-5437 tests using male Swiss CF1 mice weighing 22-27 g (N = 10 per group). · Writhing test E-mail: [email protected] All extracts displayed antinociceptive effects in the hot-plate test Presented at the XVII Annual Meeting (MET ip = 48%, MET po = 39%, CH ip = 27%, CH po = 50%, of the Federação de Sociedades de POL ip = 74%, and POL po = 49% compared to control). Pretreatment Biologia Experimental, Salvador, BA, with naloxone (2.5 mg/kg, sc) abolished the effects of CH and POL, Brazil, August 28-31, 2002. and partially prevented the analgesia induced by MET administered by the ip (but not by the po) route. POL and CH (po) significantly reduced R. Fenner, A.P. Heckler, the number of writhes induced by acetic acid, while MET was ineffec- S.M.K. Rates and A.F. Viana were the recipients of fellowships from tive in this regard. We conclude that the antinociceptive effects of the PROPESQ-UFRGS, CNPq and CAPES, H. caprifoliatum (CH) and H. polyanthemum (POL) hexane extracts respectively. seem to be mediated by the opioid system. Moreover, the antinocicep- tive activity of the H. caprifoliatum MET extract seems to depend on at least two chemical substances (or groups of substances) with distinct pharmacokinetic profiles and mechanisms of action. Only the Received April 18, 2002 naloxone-insensitive component of MET activity showed good bioa- Accepted February 17, 2003 vailability following oral administration.

The chemical investigation of the genus for the treatment of mild to moderate depres- Hypericum (Guttiferae), which comprises ap- sion (2). proximately 400 species (1), has led to the The Southern Brazilian Hypericum spe- isolation of more than 100 compounds from cies H. brasiliensis and H. connatum are about 20 species with various different bio- popularly used for relief of disorders such as logical activities, especially antiviral, anti- angina, cramps and oral and pharyngeal in- microbial and antidepressant properties. H. flammations, which suggests an analgesic perforatum extracts are widely used in Eu- property for this genus (3). rope, in the United States, and also in Brazil, Previous reports published by our group

Braz J Med Biol Res 36(5) 2003 632 A.F. Viana et al.

have shown interesting biological activities evaporated to dryness under reduced pres- for the Hypericum species native to the State sure. The extract was dissolved in saline of Rio Grande do Sul, Brazil. A crude lipo- solution containing 2.5% (w/v) polysorbate philic extract of H. caprifoliatum induces an 80. The pH of the final solutions was 6.5 to anti-immobility effect in the forced swim- 7.0. The volume administered was 1 ml/100 ming test (4), which is considered to indicate g body weight for the analgesic tests. an antidepressant action (5), as well as an Male Swiss CF1 mice (22-27 g) from the antinociceptive effect in the hot-plate test breeding colony of Fundação Estadual de (6). H. caprifoliatum, H. piriai and H. poly- Pesquisa e Ensino em Saúde (FEPPS, RS, anthemum showed in vitro monoamine oxi- Brazil) were used. The animals were housed dase A-inhibitory activity (7). The aim of the in plastic cages, 5 to a cage, under a 12-h present study was to investigate further the light/dark cycle (lights on at 7:00 h) at con- antinociceptive effects of H. caprifoliatum stant temperature (23 ± 1ºC), with free ac- and to start the characterization of the anti- cess to standard certified rodent diet and tap nociceptive properties of H. polyanthemum. water. The experiments were performed ac- Air-dried and powdered aerial parts of H. cording to the guidelines of the National caprifoliatum and H. polyanthemum were Ethics Committee on Research, Brazilian extracted with cyclohexane using an ultra- National Health Council. Ten mice per group turrax apparatus (3 x 5 min; plant/solvent were used for all experiments. ratio 1:10, w/v), yielding extracts termed CH Before actual testing on the hot plate, the and POL, respectively. In order to obtain an mice were habituated to the nonfunctioning extract rich in polar substances, H. caprifo- apparatus for 1 min. Thirty minutes later, the liatum was also extracted consecutively in a animals were placed on the functioning hot Soxhlet apparatus with petroleum ether, chlo- plate (Ugo Basile, Comerino, Italy) to deter- roform and methanol (MET) and only the mine baseline responsiveness 10 min before MET extract was used. All solvents were treatment with 90 mg/kg CH or MET or 180

Table 1. Antincociceptive effect of cyclohexane (CH) and methanol (MET) extracts of the aerial parts of Hypericum caprifoliatum and of the cyclohexane extract of aerial parts of H. polyanthemum (POL) in mice submitted to the hot-plate test.

Treatment (administration) Latency (s) % Analgesia

Before treatment After treatment

Control (saline + 2.5% polysorbate 80) 12.1 ± 0.8 12.0 ± 1.4 0 MOR (6 mg/kg, sc) 10.2 ± 1.2 27.9 ± 1.9* 100 MET (90 mg/kg, ip) 12.0 ± 1.8 20.5 ± 3.7* 48.4 CH (90 mg/kg, ip) 9.4 ± 1.5 14.2 ± 1.8* 27.3 POL (180 mg/kg, ip) 10.2 ± 1.8 23.1 ± 3.9* 73.8 NAL (2.5 mg/kg, sc) + MOR (6 mg/kg, sc) 10.3 ± 1.3 10.8 ± 1.5 3.1 NAL (2.5 mg/kg, sc) + CH (90 mg/kg, ip) 11.4 ± 1.3 11.2 ± 2.0 0 NAL (2.5 mg/kg, sc) + MET (90 mg/kg, ip) 9.7 ± 1.4 15.6 ± 2.4* 30.8 NAL (2.5 mg/kg, sc) + POL (180 mg/kg, ip) 12.37 ± 1.3 12.6 ± 3.1 1.5 CH (90 mg/kg, po) 9.2 ± 0.9 17.8 ± 1.7* 50 MET (90 mg/kg, po) 10.5 ± 0.8 17.4 ± 2.1* 39.2 POL (180 mg/kg, po) 12.26 ± 1.8 20.7 ± 2.5* 48.6 NAL (2.5 mg/kg, sc) + CH (90 mg/kg, po) 12.8 ± 1.5 11.1 ± 0.6 0 NAL (2.5 mg/kg, sc) + MET (90 mg/kg, po) 10.2 ± 1.0 16.6 ± 2.7* 36.5

Data are reported as mean ± SEM. MOR: morphine; NAL: naloxone. *P<0.005 compared to the latency of the same mouse before treatment (paired Student t-test).

Braz J Med Biol Res 36(5) 2003 Antinociceptive activity of Hypericum spp 633 mg/kg POL (ip and po). Treatment-induced revealed that none of the extracts caused any changes in responsiveness to the hot plate signs of pain or writhes per se, in mice, when were determined 30 and 45 min after ip and injected ip (8). The results obtained in the po administration, respectively. The nega- writhing test are reported as median values tive control group received an equal volume and their respective interquartile intervals, of vehicle (saline + 2.5% (w/v) polysorbate and were analyzed by the Kruskal-Wallis 80). Morphine (6 mg/kg, sc) was adminis- test. tered to the positive control group. To deter- All extracts displayed antinociceptive mine the possible involvement of opioid- effects in the hot-plate test (Table 1). Pre- mediated mechanisms, some groups of ani- treatment with naloxone abolished the ef- mals were pretreated with naloxone (2.5 mg/ fects of CH and POL, indicating that these kg, sc), a nonspecific opioid receptor an- effects are produced by opioid-mediated tagonist, immediately after evaluating base- mechanisms. Conversely, antinociception line responsiveness, 10 min before extract produced by MET administered ip was only administration. partially prevented by naloxone, whereas For the hot-plate test, mice were placed the po antinociceptive activity was not modi- on a metal surface kept at 53 ± 1ºC. The time fied, indicating that opioid-like substances elapsed until the animal licked one of its present in this extract were not absorbed by hind paws or jumped was recorded (latency the gastrointestinal tract or suffered single- time, in s) and considered to be the reaction pass inactivation by the liver. In addition, the time in both exposures. Mice that presented percent of analgesia was higher when MET baseline reaction times of more than 15 s in was administered by the ip route compared the first session were not used. In the second to the po route. session, a maximum latency time of 30 s was Administration of CH and POL signifi- imposed in order to avoid tissue damage. cantly reduced the number of abdominal The data were analyzed by the paired writhes induced by acetic acid, whereas MET Student t-test, considering the animal as its did not have a significant effect (Table 2). own control (second measure vs first meas- Interestingly, the magnitude of the antinoci- ure). The results obtained in the hot-plate ceptive effect of CH in the writhing test (oral test are reported as the mean ± SEM absolute route) was similar to that observed in the latency time or as the percent of antinocicep- tive effect relative to morphine (6 mg/kg, sc) according to the following formula: % anal- Table 2. Analgesic effects of methanol (MET) and cyclohexane (CH) extracts of aerial parts of Hypericum caprifoliatum (90 mg/kg, po) and of the cyclohexane extract of gesia = (testafter - testbefore)/(morphineafter - aerial parts of H. polyanthemum (POL, 180 mg/kg, po) on writhing induced by 0.8% morphinebefore) x 100. acetic acid (ip) in mice. The animals were treated with CH, MET # (90 mg/kg, po) or POL (180 mg/kg, po) for Treatment Number of writhes % Reduction of abdominal writhing compared to control the writhing test 45 min before receiving an ip injection of 0.8% acetic acid. Mice were Control 58 (55-65) - then placed individually in glass observation DIP 0 (2-16)* 100 MOR 0 (0-3)* 100 chambers and the number of abdominal CH 24 (0-34.5)* 58.6 writhes was counted over a period of 15 min. MET 40.5 (16-59) 30.2 The control group received an equal volume POL 0 (0-10)* 100 of vehicle (saline + 2.5% (w/v) polysorbate Control (saline + 2.5% polysorbate 80); DIP (dipyrone, 150 mg/kg, po); MOR (mor- 80, po). Dipyrone (150 mg/kg, po) was the phine, 10 mg/kg, po). positive control treatment. Previous experi- #Values are reported as medians (interquartile intervals). ments carried out in our laboratory have *P<0.001 compared to control (Kruskal-Wallis, H = 30.235).

Braz J Med Biol Res 36(5) 2003 634 A.F. Viana et al.

hot-plate test, while the effect of POL was opioid systems. None of the extracts (CH, more pronounced in the writhing test. Thus, POL or MET) contains hypericin (11). the antinociceptive properties of POL might In conclusion, extracts obtained from both be due to actions on both central and periph- species, H. caprifoliatum and H. polyanthe- eral pain systems. Apparently, the antinoci- mum, contain compounds with substantial ceptive activity is not correlated with the antinociceptive properties related, at least in antidepressant activity previously reported. part, to activation of opioid-mediated Although lipophilic extracts of H. caprifo- mechanisms. Further studies are in progress liatum were active in the Porsolt test (1,6), in order to elucidate the mechanisms under- the same was not true for MET or POL (4,7). lying the antinociceptive effects of these The CH extract is rich in phloroglucinol species. (7) and the POL extract contains benzopyrans as its main constituent (9). Phloroglucinol Acknowledgments derivatives may be responsible for the opioid- like effect since Simmen et al. (10) have The authors are indebted to Dr. Sérgio reported that hyperforin - a phloroglucinol Bordignon, Curso de Ciências Biológicas, isolated from H. perforatum - inhibited bind- Centro Universitário La Salle, for collecting ing to opioid receptors. With respect to the and identifying the plant material, and to Dr. benzopyrans as well as the flavonoid deriva- Gilsane Lino von Poser’s group, Programa tives, which are present in MET (Dall’Agnol de Pós-Graduação em Ciências Farmacêuti- R, Ferraz A, Schapoval ES and von Poser G, cas, Universidade Federal do Rio Grande do unpublished data), we are unaware of any Sul, for helping with the extract preparation. previous reports on their influence/action on

References

1. Robson NKB (1990). Studies in the genus Hypericum L. (Guttiferae) Reunião Latino-Americana de Fitoquímica, Gramado, RS, Brazil, Sep- 8. Section 29. Brathys (Part 2) and 30. Trigynobrathys. Bulletin of the tember 1999, 175. British Museum (Botany), 20: 1-151. 7. Gnerre C, Von Poser GL, Ferraz A, Viana AF, Testa B & Rates SMK 2. Barnes J, Anderson LA & Phillipson D (2001). St John’s wort (Hyperi- (2001). Monoamine oxidase inhibitory activity of some Hypericum cum perforatum L.): a review of its chemistry, pharmacology and species native to South Brazil. Journal of Pharmacy and Pharmacolo- clinical properties. Journal of Pharmacy and Pharmacology, 53: 583- gy, 53: 1273-1279. 600. 8. Viana AF (2002). Estudo da atividade psicofarmacológica de espécies 3. Mentz LA, Lutzemberger LC & Schenkel EP (1997). Da flora medici- de Hypericum nativas do Rio Grande do Sul e toxicidade aguda de nal do Rio Grande do Sul: notas sobre a obra de D’Avila (1910). Hypericum caprifoliatum Cham. & Schledt. Master’s thesis, Univer- Cadernos de Farmácia, 15: 25-47. sidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. 4. Daudt R, Von Poser GL, Neves G & Rates SMK (2000). Screening for 9. Ferraz ABF, Bordignon SAL, Staats C, Schripsema J & Von Poser GL the antidepressant activity of some species of Hypericum from (2001). Benzopyrans from Hypericum polyanthemum. Phytochemis- South Brazil. Phytotherapy Research, 14: 344-346. try, 57: 1227-1230. 5. Porsolt RD, Anton G & Blavet N (1978). Behavioral despair in rats: a 10. Simmen U, Higelin J, Berger-Büter K, Schaffner W & Lundstrom K new model sensitive to antidepressant treatments. European Jour- (2001). Neurochemical studies with St. John’s wort in vitro. Pharma- nal of Pharmacology, 47: 379-381. copsychiatry, 34 (Suppl 1): s137-s142. 6. Viana AF, Sória A, Ferraz A, Daudt R, Bordignon S, Von Poser GL & 11. Ferraz A, Bordignon S, Mans D, Schmitt A & Ravazzolo AP (2002). Rates SMK (1999). Atividade antidepressiva e analgésica de Hyperi- Screening for the presence of hypericins in southern Brazilian spe- cum caprifoliatum Cham. & Schledt (Guttiferae). In: Henriques AT cies of Hypericum (Guttiferae). Pharmaceutical Biology, 40: 294- (Editor), IX Simpósio Latino-Americano de Farmacobotânica e III 297.

Braz J Med Biol Res 36(5) 2003 Neuropharmacology 49 (2005) 1042e1052 www.elsevier.com/locate/neuropharm

The antidepressant-like effect of Hypericum caprifoliatum Cham & Schlecht (Guttiferae) on forced swimming test results from an inhibition of neuronal monoamine uptake

Alice Viana a,b, Jean-Claude do Rego b, Gilsane von Poser a, Alexandre Ferraz a, Ana Paula Heckler a, Jean Costentin b,*, Stela Maris Kuze Rates a

a Programa de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, Universidade Federal do Rio Grande do Sul. Av. Ipiranga, 2752 Porto Alegre, RS, Brazil b Unite´ de Neuropsychopharmacologie Expe´rimentale F.R.E. 2735 C.N.R.S., I.F.R.M.P. n  23, Faculte´ de Me´decine & Pharmacie, 22 Boulevard Gambetta, 76183 Rouen Cedex 03, France

Received 9 February 2005; received in revised form 27 May 2005; accepted 6 June 2005

Abstract

A crude (ECH) and a purified cyclohexane extract (HCP) of Hypericum caprifoliatum and their main phloroglucinol derivative (HC1) were evaluated regarding their action on monoaminergic systems, more precisely on dopamine. In rats and mice forced swimming test, ECH and HCP dose-dependently reduced the immobility time. The effect of the highest dose was prevented by a prior administration of either sulpiride or SCH 23390 (D2 and D1 dopamine receptor antagonist, respectively). HCP (360 mg/kg) decreased the locomotor activity of mice. ECH (90 mg/kg) caused hypothermia and potentiated apomorphine-induced (16 mg/kg) hypothermia in mice. HCP and HC1 inhibited, in a concentration-dependent and monophasic manner, the [3H]-DA, [3H]-NA and [3H]-5HT synaptosomal uptakes, but did not prevent the binding of specific ligands to the monoamine transporters. Moreover, 3 3 when tested at the concentrations corresponding to its IC50 on [ H]-DA uptake, HC1 did not induce a significant [ H]-DA release, while at a higher concentration (200 ng/ml) it enhanced significantly (by 12%) the synaptosomal DA release. These data suggest that the antidepressant-like effect of H. caprifoliatum on the forced swimming test is due to an increase in monoaminergic transmission, resulting from monoamine uptake inhibition, more potently of dopamine, which may be related to their phloroglucinol contents. Ó 2005 Elsevier Ltd. All rights reserved.

Keywords: Depression; Monoaminergic system; Forced swimming test; Hypericum caprifoliatum; Phloroglucinol derivatives

1. Introduction compounds from about 20 species, with various bi- ological activities, which are primarily antiviral, antimi- Hypericum (Guttiferae) is a large genus of herbaceous crobial and antidepressant (Wu et al., 1998). Hypericum or shrubby plants, which widely occurs in temperate perforatum extracts are used in Europe, USA, as well as regions of the world. This genus encompasses approx- in South America, for the treatment of mild to moderate imately 400 species (No¨r et al., 2004). Chemical depression. A substantial amount of experimental and investigation has led to the isolation of more than 100 clinical data demonstrate that this plant is an effective antidepressant (Linde et al., 1996; Butterweck et al., 1997; Mu¨ller et al., 1998; Mu¨ller, 2003). However, * Corresponding author. Tel.: C33 235148602; fax: C33 235148603. neither the mechanisms of action nor the identity of the E-mail address: [email protected] (J. Costentin). active constituents are completely understood. The

0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.06.002 A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052 1043 naphthodianthrone hypericin has been considered as the (FST) does not appear to be related to the MAOI most active constituent of H. perforatum (Butterweck activity, since the extracts which displayed this activity et al., 1997). In fact, some reports still provide evidences were not active in the FST (Gnerre et al., 2001). After for the role of hypericin in the antidepressant activity of phytochemical investigation of eight species, including H. perforatum (Butterweck et al., 1998) and the majority H. caprifoliatum, Ferraz et al. (2002) verified the absence of the phytomedicines available are standardized on this of hypericin in all species. Recent study, carried out by naphthodianthrone. Nevertheless, nowadays most re- No¨r et al. (2004), has shown that the phloroglucinol searchers consider that the antidepressant effects are derivatives from Hypericum species, native to southern due to a variety of constituents rather than a single one Brazil, have a dimeric structure consisting of filicinic (Chatterjee et al., 1998a; Butterweck et al., 2000). acid and phloroglucinol moiety. This molecular feature Xanthones (Rocha et al., 1994), flavonoids (Butterweck has been proposed as chemotaxonomic marker for the et al., 2000), and especially hyperforin (Chatterjee et al., species of Hypericum, and differs from hyperforin and 1998b; Mu¨ller et al., 2001), a phloroglucinol derivative, adhyperforin, which are polyisoprenylated phlorogluci- are also considered as pharmacologically relevant nol derivatives. By means of bioassay-guided fraction- compounds. ation through forced swimming test, it was concluded H. perforatum mechanism of action is still unknown, that the active substance(s) are present only in the most whereas early studies report that hypericin inhibits lipophilic (light petroleum) fraction, which primarily MAO in the concentration range of 50 mg/ml (Suzuki consists of phloroglucinol derivatives (Daudt et al., et al., 1984), other authors have failed to confirm this 2000). This class of secondary metabolites has been effect (Yu, 2000). In fact, the inhibitory activity was reported as being one of the main compounds re- reached at concentrations considered too high sponsible for H. perforatum activity, in pre-clinical as (Ki Z 2 mg/ml for MAOA and 3.2 mg/ml for MAOB)to well as clinical assays (Chatterjee et al., 1998a; Mu¨ller, be therapeutically relevant (Suzuki et al., 1984). 2003). Presently, several reports have showed that the main The aim of this study was to further investigate the therapeutically used H. perforatum extract (LI 160, H. caprifoliatum antidepressant-like effect in order to standardized at 0.3% of hypericin) is a potent but evaluate its mechanism of action. Therefore, after unspecific inhibitor of the neuronal uptake of several attempting to address the in vivo monoaminergic effects biogenic amines and amino acid neurotransmitters of H. caprifoliatum purified lipophilic extracts on exper- (Mu¨ller et al., 1998). Currently, some reports have imental model of depression, the forced swimming test, we attempted to link this nonselective profile to an effect of attempted to assess their effects on the dopaminergic, hyperforin on sodium channels (Singer et al., 1999; noradrenergic and serotoninergic neuronal systems. Mu¨ller et al., 2001). In agreement with this hypothesis, Buchholzer et al. (2002) have demonstrated that hyper- forin inhibits the sodium dependent high-affinity choline 2. Materials and methods uptake and thereby the striatal acetylcholine release. Furthermore, Roz and Rehavi (2003) have reported that 2.1. Plant material the effect of hyperforin on the uptake of monoamines could be explained by its action on the pH gradient in The aerial parts of H. caprifoliatum were collected in the synaptic vesicle membrane. the region of Viama˜ o, in the state of Rio Grande do In Southern Brazil, 20 Hypericum species have been Sul e Brazil (August/2001). The voucher specimens identified (Robson, 1990). However, scientific reports were deposited in the herbarium of the Federal regarding these species are scarce. Xanthones, flavo- University of Rio Grande do Sul (ICN) (Bordignon noids and phloroglucinols were isolated from Hyper- 1496). icum brasiliense and its antimicrobial and monoamine oxidase inhibitory activities were demonstrated by 2.2. Preparation of extracts Rocha et al. (1994, 1995). Schmitt et al. (2001) have reported an antiviral activity in vitro against feline The dried and powdered plant material (120 g of immunodeficiency virus (FIV) of Hypericum connatum. aerial parts) was extracted with cyclohexane (plant/ As regards a possible antidepressant effect, Hyper- solvent ratio 1:10 w/v) by turbo-extraction during 5 min icum caprifoliatum showed promising results in the followed by evaporation to dryness under reduced forced swimming test, which predicts antidepressant pressure at 45 C yielding an extract termed ECH (ca. activity (Daudt et al., 2000). The in vitro monoamine 5.0 g). To obtain the purified extract, ECH was treated oxidase inhibitory (MAOI) activity of the extracts of H. with acetone, according to Rocha et al. (1995), pro- caprifoliatum and some other Brazilian species has also ducing an insoluble fatty residue (10% w/w), which was been demonstrated (Gnerre et al., 2001). Nevertheless, eliminated through a paper filter. Then, chlorophyll was the effect of H. caprifoliatum in the forced swimming test removed by mixing the acetone soluble extract with 1044 A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052 powdered charcoal. This mixture was filtered and the submitted to swimming for 15 min in water with solvent evaporated under reduced pressure at 45 C temperature between 23 G 2 C and height of 30 cm yielding 4.0 g of a wax and chlorophyll free extract (Porsolt et al., 1978 employed 15 cm). The ambient termed HCP. temperature was approximately 22 C. At the end of the swimming exposition, the animals were removed from 2.3. Chemical characterization of the extracts the water and gently dried. The treatment was admin- istered 5 min, 19 and 23 h after the first swimming The ECH and HCP were analyzed by preparative exposition; the first administration was carried out Thin Layer Chromatography (TLC) using silica gel between 2:00 and 5:00 p.m.; the second, between 9:00 GF254 as stationary phase, chloroform/hexane (1:1 v/v) and 12:00 a.m. and the third, between 1:00 and 4:00 p.m. as mobile phase and Godin’s reagent as chromogenic One hour after the last injection (24 h after the first agent characteristic for phloroglucinol derivatives (Ro- swimming session), the animals were submitted to cha et al., 1995). Both extracts showed a main spot (Rf a second swimming exposure (5 min), and their immo- 0.10e0.40), reactive to Godin’s reagent, which was bility time was measured. removed by preparative TLC (90 mg) and characterized In preliminary doseeresponse studies, we observed by NMR (1H and 13C, 400 MHz). This compound was that ECH 270 mg/kg/day, p.o. (three administrations of termed HC1. All solvents (pro-analysis) were obtained 90 mg/kg), afforded the maximal anti-immobility effect from MERCK Kga (Darmstadt, Germany). in rats (data not shown). Thus, this dose was selected for continuing the study. The ECH and HCP were diluted 2.4. Behavioral experiments to a concentration of 90 mg/ml (270 mg/kg/day, p.o.) in water with 2% of polysorbate 80 or 10% DMSO, 2.4.1. Animals respectively. Imipramine hydrochloride 20 mg/kg (60 mg/ Adult male Wistar rats (weight 200e300 g) and male kg/day, p.o.) was used as the antidepressant reference CF1 Swiss mice (25e30 g) purchased from Fundac¸a˜ o drug and 2% of polysorbate 80 or 10% DMSO in saline Estadual de Produc¸a˜ o e Pesquisa em Sau´de e RS were used as the negative control. For evaluating the (Brazil) colony were used. The animals were housed 5 dopaminergic action of the extracts, the animals were rats or 20 mice in plastic cages (L: 42 cm, W: 27.5 cm, H: treated with sulpiride (50 mg/kg, i.p.) 30 min prior to the 16 cm). All animals were kept under a 12-h light/dark last administration of the solutions to be tested. In this cycle (lights on at 7:00 a.m.) at a constant temperature case, bupropion was used at the 20 mg/kg dose (60 mg/ of 23 G 1 C with free access to standard certified kg/day, p.o.) as the dopaminergic antidepressant refer- rodent diet (Nuvilab CR-1) and tap water. ence drug. All treatments were administered at 1 ml/kg All the behavioral experiments were approved by body weight. CONEP e Brazil (National Commission of Research Ethics) and performed according to the guidelines of 2.4.3.2. In mice. The apparatus consisted of a glass The National Research Ethical Committee (published cylinder (10 cm internal diameter, 25 cm height) filled by National Heath Council e MS, 1998), which are in with water (19 cm height) at 23 G 2 C. The HCP was compliance with the European Communities Council diluted to concentrations between 9 and 36 mg/ml in Directive of 24 November 1986 (86/609/EEC). water with 2% of polysorbate 80. Different groups of mice were treated with HCP (90, 180, 270 or 360 mg/kg, 2.4.2. Drugs p.o.), imipramine (20 mg/kg, p.o.), bupropion (30 mg/ Bupropion HCl (Glaxo Wellcome, Sa˜ o Paulo, Brazil), kg, p.o.), or 2% polysorbate 80 solution in water. For imipramine HCl (Galena, Sa˜ o Paulo, Brazil), sulpiride evaluating the dopaminergic action, the different groups (DEG, Sa˜ o Paulo, Brazil), apomorphine (Sigma, St. were pre-treated with sulpiride (50 mg/kg, i.p.) or SCH Louis, MO), dimethylsulfoxide (DMSO), cyclohexane, 23390 (15 mg/kg, s.c.) 30 min before the administration polysorbate 80 (Merck, Darmstadt, Germany), SCH of the most effective dose of HCP. All treatments were 23390 (SigmaeAldrich, Saint Quentin Fallavier, France). administered at 10 ml/kg body weight. After 60 min, the animals were placed into the cylinder and the total duration of immobility, during a 6-min test period, was 2.4.3. Forced swimming test (FST) measured. A mouse was considered immobile when it remained floating in the water, and only makes move- 2.4.3.1. In rats. The Porsolt’s procedure (Porsolt et al., ments necessary to keep its head above the water. 1978) was used with minor modifications. In this test an acrylic box with four sections of 30 ! 30 ! 40 cm was 2.4.4. Locomotor activity used. The external walls and the cover were transparent, Locomotor activity was accessed automatically in a but the inside sections were dark allowing the isolation digiscan photocell activity meter box (45 ! 30 ! 30 cm) of each one of the four quadrants. The rats were (Omnitech Electronics Inc, Columbus, OH). The A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052 1045 response to HCP 360 mg/kg, administered by gavage 2.5.3. Synaptosomal uptake of [3H]-DA, [3H]-5HT (10 ml/kg body weight) immediately before the test, and [3H]-NA was expressed as the number of beams crossed between All procedures necessary to prepare synaptosomal 5 and 45 min after treatments. suspensions were performed at 0e2 C. Animals were sacrificed by decapitation; their striata (for [3H]-DA uptake), frontal cortex (for [3H]-5HT uptake) and 2.4.5. Apomorphine-induced hypothermia hypothalamus (for [3H]-NA uptake) were dissected out Temperature measurements were performed in a and homogenized with 12 up and down strokes of G  temperature-controlled room (23 1 C) between 9:30 a Tefloneglass homogenizer (800 r.p.m.) in 10 volumes and 11:30 a.m. The mice colonic temperature was re- (w/v) of ice-cold 0.32 M sucrose solution containing corded using commercially available thermometer Ò 0.1 mM pargyline. The nuclear material was removed by (Pro´-check ), which was dipped in Vaseline and inserted centrifugation at 1000 g for 10 min, and the supernatant about 1 cm in the gently hand-restrained mouse. (crude synaptosomal fraction) was used in uptake After recording their initial colonic temperature experiments. Z (t 0) different groups of mice received either ECH Aliquots (100 ml) of the crude synaptosomal fraction (90 mg/kg, p.o.) or solvent (polysorbate 80, 2% in were preincubated for 5 min at 37 C in a KrebseRinger water) (1 ml/100 g). Thirty minutes later, all animals medium, containing (mM): NaCl 109, KH2PO4 1, CaCl2 were treated with apomorphine (16 mg/kg, s.c.). Their 1, NaHCO3 27, glucose 5.4, pH 7.4 G 0.1, in the presence colonic-temperature was recorded 15 and 30 min after or absence of HCP or HC1. The incubation was the last treatment. continued for 5 min in the same medium, in the presence of [3H]-DA, [3H]-5HT or [3H]-NA (10 nM; 1 ml final 2.5. In vitro assays volume). The reaction was stopped by adding 3 ml of ice- cold incubation medium and immediate centrifugation  2.5.1. Animals (7000 g, 10 min, 4 C). The pellet was washed with 1 ml Male SpragueeDawley rats, weighing 180e200 g, of the latter medium and centrifuged in the same were purchased from IFFA-CREDO/Charles River condition. The final pellet was sonicated in 250 ml Laboratories (Domaine des Oncins, Saint-Germain sur distilled water and aliquots of the homogenate were L’Arbresle, France). They were housed by four in used for the determination of radioactivity and protein Makrolon cages (L: 40 cm, W: 25 cm, H: 18 cm), with concentrations. The radioactivity was determined by free access to water and food (U.A.R., France) and kept liquid scintillation spectrometry (Betamatic V, KON- in a well ventilated room, at a temperature of 21 G 1 C, TRON, Trapes, France) in 4 ml of Optiphase Highsafe II e under a 12-h light/dark cycle (lights on between 7:00 with 33 36% counting efficiency. Protein concentra- hour and 19:00 hour). The procedures used in this study tions were determined according to the method of Lowry are in compliance with the European Communities et al. (1951), using bovine serum albumin as standard. Council Directive of 24 November 1986 (86/609/EEC). The specific uptake of DA, 5HT or NA was defined as the difference between the total uptake at 37 C and the non-specific accumulation observed at 0 C, 2.5.2. Drugs respectively, in the presence of 100 mM cocaine for [3H]-dopamine ([3H]-DA, 48 Ci/mmol) and [3H]- [3H]-DA, 1 mM fluoxetine for [3H]-5HT or 0.3 mM nisoxetine (86 Ci/mmol) were purchased from Amer- desipramine for [3H]-NA. [3H]-5HT and [3H]-NA sham (Les Ulis, France). [3H]-noradrenaline ([3H]-NA, uptake assays were performed in the presence of 12.5 Ci/mmol), [3H]-serotonin ([3H]-5HT, 25.5 Ci/ 30 nM 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-pro- mmol), [3H]-citalopram (84.2 Ci/mmol) and [3H]-mazin- penyl)-piperazine, dihydrochloride (GBR 12783) in dol (24.5 Ci/mmol) were purchased from Perkine order to block 5HT and NA transporters operated by ElmereNEN Life Science Products (Paris, France). DA nerve terminals present, respectively, in cortical and Cocaine hydrochloride was obtained from la Coope´ra- hypothalamic preparations. tive Pharmaceutique Franc¸aise (Melun, France). De- The monoamine synaptosomal uptake was analyzed sipramine hydrochloride, fluoxetine hydrochloride and using the equation of the sigmoidal doseeresponse GBR 12783 were purchased from SigmaeAldrich (Saint curve (variable slope). IC50 and n Hill values were Quentin Fallavier, France). Solutions of HCP and HC1 calculated using the equation of the curve-fitting extracts (10ÿ4e10ÿ11 g/ml) were prepared in an in- programs Microcal Origin (Microcal Software). cubation medium containing 5% cremophor and 5% DMSO. Millimolar solutions of GBR 12783 were prepared in distilled water. Subsequent dilutions and 2.5.4. Binding assays to monoamine transporters solutions of other agents were performed in the The binding to dopamine (DAT), noradrenaline incubation medium. (NAT) or serotonin (SERT) transporters was assessed, 1046 A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052 respectively, on striatal, cortex and hypothalamus mem- of modified KrebseRinger medium, corresponding to branes of rats. The crude synaptosomal suspensions, [3H]-DA loaded synaptosomal fraction (S2). prepared as described in Section 2.5.3, were centrifuged at To assess the [3H]-DA releasing effect of HC1 the 17,000 g for 30 min at 4 C. The pellet was resuspended procedure was performed according to Carruba et al. by sonication in: 10 mM NaC medium (0.30 mM (1977), by incubating (10 min at 37 C) 250 ml aliquots NaH2PO4, 9.70 mM NaHCO3, pH 7.5 G 0.1) for the of synaptosomal samples S2, in Eppendorf tubes binding to DAT or 50 mM TriseHCl (containing containing Ca2C free modified KrebseRinger medium, 120 mM NaCl and 5 mM KCl, pH 7.4 G 0.1) for NAT in the absence (control) or presence of HC1 (100 ng/ml and SERT and centrifuged (50,000 g, 10 min, 4 C). The or 200 ng/ml final concentrations), in a total volume of final pellet was resuspended by sonication in the same 500 ml. The releasing effect was stopped by centrifuga- respective medium for the binding to DAT and SERT, for tion (7000 g, 10 min, 4 C). Aliquots of 400 ml from the NAT it was resuspended in 50 mM TriseHCl (containing last supernatant, as well as 250 ml of synaptosomal 300 mM NaCl and 5 mM KCl, pH 7.4 G 0.1). samples S2, were measured by liquid scintillation spec- [3H]-mazindol (2 nM final concentration) and mem- trometry (Betamatic V, KONTRON, Trapes, France) in branes (100 mg protein) were incubated at 0 C, for 2 h, 4 ml of Optiphase Highsafe II (33e36% counting to evaluate the binding to DAT. For the binding to efficiency). The amount of [3H]-DA released is expressed NAT 300 mg of protein were incubated with [3H]- as percentage of the total radioactivity contained in nisoxetine (1 nM) at 25 C during 2 h; and for SERT, 250 ml synaptosomal samples S2. The values obtained 200 mg of protein were incubated with [3H]-citalopram represent the means G SEM of three independent ex- (1 nM) during 1 h at 25 C. All incubations were carried periments, each performed in triplicate. out in the presence of different HC1 concentrations 7 11 (3 ! 10ÿ e3 ! 10ÿ g/ml), in a final volume of 500 ml. 2.6. Statistical analysis Incubations were stopped by dilution with 3 ml of ice- cold incubation medium and immediate filtration The data were evaluated using one or two-way through GF/B filters, previously soaked, for at least analysis of variance (ANOVA) followed by Studente 1 h, in 0.5% polyethyleneimine. Each tube and filter was NewmaneKeul’s test or Student’s t-test depending on rinsed once or twice with 3 ml of ice-cold incubation the experimental design. p-Values less than 0.05 were medium. Filters were counted for radioactivity by liquid considered as statistically significant. scintillation spectrometry (Tri-carb 2100TR, Packard BioScience Co.) in 4 ml of Ultima Gold (Perkine Elmer). The non-specific binding was determined by 3. Results incubation with cocaine (100 mM, for DAT), desipra- mine (10 mM, for NAT) or fluoxetine (10 mM, for 3.1. Chemical characterization SERT). Protein concentrations were determined accord- ing to the method of Lowry et al. (1951), using bovine HC1 appears to be the main compound of both ECH serum albumin as standard. and HCP extracts, representing 8% of HCP. TLC and spectral data revealed that HC1 is a phloroglucinol 3 2.5.5. Synaptosomal [ H]-DA release derivative. The NMR spectra gave hydroxyl resonance The DA release was determined on synaptosomes at ca. d 18, 16, 11, and 10. The unusually low field 3 previously loaded with [ H]-DA. The striata synapto- shifted signals (ca. d 18) suggest the presence of hydroxyl somes were prepared as described in Section 2.5.3. The protons that participate in rather strong hydrogen crude synaptosomal suspension was centrifuged for bonds. This signal is frequently observed in NMR 30 min at 17,000 g and the resulting pellet was resus- spectra of phloroglucinol derivatives consisting of acyl e pended in the uptake incubation Krebs Ringer medium filicinic acid and phloroglucinol moieties linked by (described in Section 2.5.3) corresponding to the total a methylene bridge. Furthermore, the spectra revealed synaptosomal fraction (S1). An aliquot (1 ml) of S1 was several signals at ca. d 18 which could indicate a mixture e diluted in 2900 ml of Krebs Ringer and was incubated for of tautomeric forms (Verotta et al., 2000). The purifi-  3 10 min at 37 C in the presence of 100 mlof[H]-DA cation and complete structural elucidation of this com- 3 (100 nM final concentration). The [ H]-DA loading was pound are in progress. stopped by centrifugation (7000 g, 10 min, 4 C) followed by two washings of the pellet in 4 ml of Ca2C free modified KrebseRinger medium (NaCl 109 mM, KH2PO4 1 mM, 3.2. Behavioral experiments MgSO4 1.2 mM, NaHCO3 27 mM, glucose 5.4 mM, ascorbic acid 1 mM and pargyline 0.05 mM at pH 3.2.1. Forced swimming test 7.5 G 0.1) to remove radioactivity not uptaken into the Two different sets of experiments were performed. synaptosomes. The final pellet was then suspended in 4 ml The first was carried out to confirm whether the A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052 1047 immobility time reported by Daudt et al. (2000) in 300 rats treated intraperitoneally with the H. caprifoliatum petroleumeether extract, was also observed in animals 250 treated per os with the cyclohexane extract (ECH) or the * 200 * cyclohexane purified extract (HCP) of H. caprifoliatum * per os. Both ECH and HCP were effective on rat FST 150 * Z * * when compared to solvent (ANOVA F4,62 15.39, * * ! * * p 0.001) (Fig. 1). In mice, the immobility time was * Immobility (s) 100 dose-dependently reduced by HCP administration and * the highest tested dose was the most effective (360 mg/ 50 kg) (ANOVA, F6,57 Z 15.5, p ! 0.001) (Fig. 2). The second set of experiments was performed in order to 0 evaluate whether the dopaminergic system was involved CONT IMIBUP HCP90 HCP180 HCP270 HCP360 in the anti-immobility effect. The injection of sulpiride, Fig. 2. Anti-immobility effect of imipramine 20 mg/kg, p.o. (IMI), 30 min prior to the last administration of the extract bupropion 30 mg/kg, p.o. (BUP) and H. caprifoliatum purified or bupropion, impaired the immobility time reduction cyclohexane extract (HCP 90, 180, 270 and 360 mg/kg, p.o.) in mice caused by either bupropion or ECH treatment in rats forced swimming test. The results are presented in mean G SEM (two-way ANOVA, pre-treatment ! treatment factor, (n Z 8e12 mice/group). StudenteNewmaneKeul’s post hoc compar- isons: ***p ! 0.001 compared with control (CONT). F2,64 Z 12.71, p ! 0.001) (Fig. 3). The anti-immobility effect in mice was prevented by sulpiride (50 mg/kg) (two-way ANOVA, pre-treatment ! treatment factor, was 520 G 98 for HCP treated animals (Student ’s t-test, F1,33 Z 14.77, p ! 0.001) (Fig. 4) or by SCH 23390 t Z 2.24; p ! 0.05). (15 mg/kg) pre-treatment (two-away ANOVA, pre- treatment ! treatment factor, F1,30 Z 8.87, p ! 0.01) (Fig. 4). 3.2.3. Apomorphine-induced hypothermia ECH alone provoked significant hypothermia. Fur- 3.2.2. Locomotor activity thermore, in mice pre-treated with ECH, an enhance- The treatment with HCP, 360 mg/kg, p.o., significantly ment of apomorphine-induced hypothermia was Z reduced the locomotor activity of mice. The mean number observed (two-way ANOVA, time factor, F2,88 89.3; of beams crossed by control animals was 814 G 87 while it 300

300 250 c 250 c # 200 # # # # # 200 * 150 * * * *

* Immobility (s) 150 * * * * * * * 100 * *

Immobility (s) * 100 * * 50

50 0 CONT BUP ECH SUL BUP+S ECH+S

0 Fig. 3. Effect of SUL (sulpiride 50 mg/kg, i.p.) administration on the CONT IMIBUP ECH HCP anti-immobility action of ECH 270 mg/kg/day (p.o.) in rat forced swimming test. BUP Z bupropion 60 mg/kg/day, p.o., ECH Z cyclo- Fig. 1. Anti-immobility effect of imipramine 60 mg/kg/day, p.o. (IMI), hexane extract, BUP C S Z bupropion C sulpiride, ECH C S Z cy- bupropion 60 mg/kg/day, p.o. (BUP), cyclohexane extract (ECH) and clohexane extract C sulpiride. The results are presented in mean G purified ECH (HCP) of H. caprifoliatum (270 mg/kg/day, p.o.) in rat SEM (n Z 12e14 rats/group). cp ! 0.001 (Two-way ANOVA, BUP forced swimming test. The results are presented in mean G SEM or ECH ! SUL interaction). StudenteNewmaneKeul’s post hoc (n Z 12e14 rats/group). StudenteNewmaneKeul’s post hoc compar- comparisons: ***p ! 0.001 significant difference in relation to control isons: ***p ! 0.001 significant difference in relation to control (CONT). ###p ! 0.001 significant difference in relation to the re- (CONT). spective group without SUL. 1048 A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052

300 Table 2 c Comparison of the inhibition of monoamine uptake by HCP and HC1 250 # # Uptake Uptake Uptake b # of [3H]-DA of [3H]-5HT of [3H]-NA 200 # # IC50 n Hill IC50 n Hill IC50 n Hill 150 * (ng/ml) (ng/ml) (ng/ml) * * 100 HCP 98 G 18 1.0 G 0.15 520 G 32 0.8 G 0.1 971 G 98 0.74 G 0.1 Immobility (s) HC1 85 G 19 1.2 G 0.25 599 G 60 0.7 G 0.1 206 G 24 0.70 G 0.1 50 Data were calculated using the equation of the curve-fitting programs

Microcal Origin. IC50 and n Hill values represent the mean G SEM of 0 four independent experiments, carried out in duplicate. CONT HCP SCH HCP+SCH SUL HCP+SUL

Fig. 4. Effect of SCH 23390 (15 mg/kg, s.c.) or SUL (sulpiride 50 mg/ 3 kg, i.p.) administration on the anti-immobility action of HCP (H. effective against [ H]-5HT and 2.5 (HC1) and 10 3 3 caprifoliatum cyclohexane extract, 360 mg/kg, p.o.) in mice forced (HCP) times less effective on [ H]-NA than on [ H]- swimming test. The drugs were injected 30 min before the extract DA uptake. Nevertheless, no significant difference was treatment. The results are presented in mean G SEM (n Z 8e12 mice/ 3 3 found between the IC50 [ H]-DA and [ H]-5HT uptake b ! c ! ! group). p 0.01; p 0.001 (Two-way ANOVA, HCP SCH values of HCP and HC1. In contrast, concerning the interaction or HCP ! SUL interaction). ***p ! 0.001 compared with 3 control (CONT). ##p ! 0.01; ###p ! 0.001 compared with HCP. [ H]-NA uptake, HC1 was more effective, with IC50 value about five times lower than the IC50 of HCP. treatment factor, F Z 23.3; time ! treatment factor, 3,88 3.3.2. Effects of HC1 on the binding of uptake F Z 14.75, p ! 0.001 for all factors) (Table 1). 6,88 inhibitors to monoamine transporters The different tested concentrations of HC1 did not 3.3. Neurochemical experiments inhibit, in a statistically significant manner, the binding of [3H]-mazindol, [3H]-nisoxetine, [3H]-citalopram to 3.3.1. Effects of HCP and HC1 on the uptake their respective monoamine transporters (Table 3). of biogenic amines 3 3 HCP and HC1 inhibited [ H]-DA, [ H]-5HT and 3.3.3. Effects of HC1 on synaptosomal 3 [ H]-NA uptake by crude synaptosomal suspensions [3H]-DA release prepared from rat striatum (for DA), rat frontal cortex The level of spontaneous [3H]-DA release by synap- (for 5HT) and hypothalamus (for NA), in a concentra- tosomes, previously loaded with [3H]-DA and incubated 2C tion-dependent and monophasic manner. The IC50 in Ca free KrebseRinger medium, was not signifi- values with respective Hill coefficients (n Hill), which cantly modified in the presence of 100 ng/ml HC1. In were close to unity, are summarized in Table 2. It should contrast, at the 200 ng/ml concentration, HC1 increased be pointed out that HCP and HC1 potently inhibited significantly, by about 12% (ANOVA, F2,8 Z 16.66, 3 Z G G 3 [ H]-DA uptake (IC50 98 18 ng/ml and 85 19 ng/ p ! 0.05) the [ H]-DA release. The percentages of ml), being, respectively, five and seven times less [3H]-DA released by pre-loaded synaptosomes were 31.5 G 1.6% in absence (control), 38 G 2.4% in the Table 1 Effect of the cyclohexane extract of aerial parts of Hypericum Table 3 caprifoliatum (ECH) (90 mg/kg, p.o.) on apomorphine-induced hypo- Effect of HC1 on the binding of [3H]-mazindol, [3H]-nisoxetine and thermia (16 mg/kg, i.p.) [3H]-citalopram  Treatments Temperatures ( C) Concentration Binding of Binding of Binding of t Z 0 t Z 15 min t Z 30 min of HC1 [3H]-mazindol [3H]-nisoxetine [3H]-citalopram (g/ml) (fmol/mg prot) (fmol/mg prot) (fmol/mg prot) SAL 38.2 G 0.1 37.8 G 0.1 38 G 0.1 APO 38.1 G 0.1 35.8 G 0.25***### 35 G 0.3***### 0 2834 G 452 48 G 2 243 G 15 ECH 38.3 G 0.5 37.5 G 0.3## 37.2 G 0.3## 3 ! 10ÿ7 3120 G 418 49 G 2 246 G 18 ECH C APO 38.1 G 0.15 34.9 G 0.35***###a 33.8 G 0.3***###b 10ÿ7 3110 G 431 52 G 3 250 G 18 3 ! 10ÿ8 2918 G 370 54 G 4 245 G 17 SAL Z saline; APO Z apomorphine 16 mg/kg; ECH Z cyclohexane 10ÿ8 3026 G 465 53 G 3 248 G 15 extract 90 mg/kg; ECH C APO Z cyclohexane extract 90 mg/kg 3 ! 10ÿ9 2901 G 391 53 G 4 252 G 15 followed by apomorphine 16 mg/kg. The results are presented in 10ÿ9 2815 G 397 53 G 3 242 G 19 mean G SEM of 8e15 mice/group.Two-way ANOVA, time factor 3 ! 10ÿ10 2785 G 390 51 G 3 253 G 23 F Z 89.3; treatment factor F Z 23.3; time ! treatment factor 2,88 3,88 10ÿ10 2794 G 396 50 G 4 243 G 17 F Z 14.75, p ! 0.001 for all factors. ***p ! 0.001 significant 6,88 3 ! 10ÿ11 2885 G 437 49 G 2 240 G 17 difference in relation to saline. ##p ! 0.01, ###p ! 0.001 significant difference in relation to t Z 0 within the group. ap ! 0.05, bp ! 0.01 Mean G SEM of four (for [3H]-nisoxetine and [3H]-citalopram significant difference in relation to apomorphine within the same time. binding) and six (for [3H]-mazindol binding) different experiments. A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052 1049 presence of 100 ng/mg HC1 and 43.2 G 3.2% in the role of D1 receptors are more controversial. D1 presence of 200 ng/ml HC1. dopamine agonists have been reported to be effective in animal models of depression by some authors (D’Aquila et al., 1994; Gambarana et al., 1995) but 4. Discussion not by others (Duterte-Boucher et al., 1988; Geofroy and Christensen, 1993). In contrast, functional interac- The results of our study demonstrate that the tion between D1 and D2 receptors has been well antidepressant-like effect of H. caprifoliatum petroleum documented in various behaviors, they are known to light crude extract, previously observed in rats by interact synergistically in some behaviors (Menon et al., Daudt et al. (2000), is reproduced by other lipophilic 1988). Furthermore, SCH 23390, in addition to being and purified extracts rich in phloroglucinols. We also a highly potent D1 receptor antagonist, binds with observed that this effect is reproducible since it was a high-affinity to 5HT2 and 5HT1C receptors (Bourne, dose-dependently reproduced when the same extracts 2001); it is also effective in blocking 8-OH-DPAT were evaluated on mice FST. Furthermore, this anti- (5HT1A agonist) effects in FST (Luscombe et al., immobility effect was not related to a non-specific 1993). It would be possible to postulate that 5HT plays behavioral stimulation, given that HCP reduced motor a role in the effect of H. caprifoliatum in the FST, despite activity. This observation supports the hypothesis that, the fact that this test frequently fails to detect to be considered as a potential antidepressant, a drug antidepressant action of serotonergic drugs (Porsolt must reduce immobility in FST at doses that do not et al., 1991). However, our results demonstrate that stimulate locomotion (Porsolt et al., 1978). This is in HCP and its main phloroglucinol rich extract (HC1) agreement with previously reported studies, which show more potently inhibit the DA synaptosomal uptake than that many antidepressants tend to decrease motor those of NA and 5-HT, confirming involvement of the activity (Duterte-Boucher et al., 1988; Bourin, 1990). dopaminergic transmission in the antidepressant-like Moreover, we also observed that ECH enhanced the effect of H. caprifoliatum lipophilic extracts (ECH, hypothermic effect of apomorphine 16 mg/kg and HCP). It appears that these effects are related to the displayed a mild intrinsic hypothermic effect. This result phloroglucinol derivative present in HC1 since, al- was unexpected because the hypothermia induced by though this fraction constitutes only 8% of HCP, it a high dose of apomorphine is strongly antagonized by inhibited [3H]-DA synaptosomal uptake with potency antidepressants such as imipramine-like drugs (Puech equivalent to HCP. Contrary to all other known et al., 1978; Menon et al., 1988). antidepressant drugs, the inhibition of monoamine Puech et al. (1981) suggested that apomorphine- uptake operated by H. caprifoliatum extracts is not induced hypothermia results from two effects: (i) the first, due to a binding of HC1 or a blockage of DA, NA or 5- observed at small doses and antagonized by neuroleptics, HT transporters at the binding sites of [3H]-mazindol, 3 3 is related to stimulation of D2 dopaminergic receptor and [ H]-nisoxetine, [ H]-citalopram, respectively. These (ii) the second, induced by high doses and antagonized by observations suggest that the mechanism of action of imipramine, is probably not related to dopaminergic H. caprifoliatum extract is probably not associated with system, but to the b-adrenergic system. Furthermore, a specific binding on each of the different monoamine DA agonists are known to be effective on FST (Duterte- transporters, but could depend on mechanisms involved Boucher et al., 1988) as well as in depressed patients (Post in neurotransmitter transport in general. This assump- et al., 1978). These results prompted us to evaluate the tion had been previously reported for other species of involvement of the dopaminergic system in the ECH and Hypericum, such as H. perforatum (Chatterjee et al., HCP effects on the FST. 1998b; Mu¨ller et al., 1997, 1998) and H. triquetrifolium The anti-immobility effects of ECH and HCP were (Roz et al., 2002). In these reports, several in vivo and in entirely prevented by sulpiride (D2 antagonist) and vitro animal studies have demonstrated that the partially prevented by SCH 23390 (D1 antagonist), at antidepressant properties and the inhibitory effects of doses devoid of locomotor impairment (Barghon et al., the extracts from these species on the neuronal uptake of 1981; Vaugeois et al., 1996), suggesting that the anti- the monoamines were due to the lipophilic phloroglu- immobility effect of H. caprifoliatum is mediated by D2 cinol derivative, hyperforin (Chatterjee et al., 1998a,b; and possibly by D1 dopamine receptor activation. Mu¨ller et al., 1998; Jensen et al., 2001; Roz et al., 2002), Nevertheless, the impairment caused by SCH 23390 on which is considered as the main active substance of H. the effect of HCP was puzzling, since Vaugeois et al. perforatum and H. triquetrifolium. These studies have (1996) have demonstrated that effects of indirect DA considered that hyperforin operates in a non-specific agonists on the despair test depend on the stimulation manner since it inhibits the uptake of each biogenic of D2 but not D1 dopamine receptors. In fact, the amine with an almost identical potency (Chatterjee effectiveness of D2 receptor stimulation on the anti- et al., 1998b; Mu¨ller et al., 1998; Roz et al., 2002) that immobility effect in FST is generally accepted, while the extends to GABA and L-glutamate uptakes (Chatterjee 1050 A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052 et al., 1998b; Singer et al., 1999; Wonnemann et al., technique in the prefrontal cortex of awake rats, 2000). In contrast, HCP and HC1 were more effective on Yoshitake et al. (2004) showed that a single dose of DA than NA and 5-HT accumulation in synaptosomes. hydro-alcoholic extracts of H. perforatum (60 mg/kg i.p. However, analyzing the data of Mu¨ller et al. (1997, or 300 mg/kg p.o.) caused: a marked increase in the 1998) and Chatterjee et al. (1998b), it appears that the extracellular levels of DA (165 and 140%, respectively); difference in the IC50 values to the uptake inhibition of a moderate increase in the extracellular level of 5HT DA are 3e5 times lower than IC50 values to NA and (about 135%); whereas NA levels remained unchanged. 5-HT uptake inhibition, which is statistically different. Based on the present study and other studies (Roz et al., Thus, in these studies, the phloroglucinol rich extract 2002; Gobbi et al., 1999; Yoshitake et al., 2004) the low from Hypericum species behaves like an antidepressant magnitude though significant efflux of [3H]-monoamines with a preferential impact on the dopaminergic system, triggered by phloroglucinol rich extracts is probably due which was not considered. In addition, although the to diffusion of the monoamines across the plasmatic 3 IC50 for [ H]-DA uptake inhibition of HCP and HC1 membrane following their concentration gradient. is approximately twice as high as that of hyperforin and In conclusion, the results obtained in our study are in hyperforin enriched CO2 extract (Chatterjee et al., accordance with previous reports regarding the effect 1998b), H. caprifoliatum appears to be in the same of lipophilic extracts of H. caprifoliatum in the forced range of potency as other Hypericum species in the FST. swimming test (FST) (Daudt et al., 2000; Gnerre et al., In order to obtain an effect similar to imipramine 30 mg/ 2001), an animal model for selecting antidepressant kg/day in the rat FST, a dose of 270 mg/kg/day of ECH drugs with a good predictive value (Porsolt et al., 1991). and HCP were required, while the effective doses of They suggest a mechanism of action that could be alcoholic extracts from other species ranged from 300 related to the inhibition of neuronal monoamine uptake, to 3000 mg/kg/day in rats (Butterweck et al., 1997; most potently of dopamine. This effect could be caused Chatterjee et al., 1998a). In the mice FST, the effective by an indirect effect of HC1 on monoamine trans- doses of other Hypericum extracts are between 250 porters, most likely due to the elevated neurotransmitter and 1000 mg/kg (O¨ztu¨rk et al., 1996; Sanchez-Mateo levels in the synaptic clefts, thus leading to an alteration et al., 2002), for HCP the lowest effective dose, which of monoamine transporter function different from the promotes an effect almost equivalent to imipramine mechanism of action of reference monoamine uptake 20 mg/kg is 270 mg/kg. inhibitors. Our further investigation are focused on The molecular mechanisms by which HC1 or other evaluating the effects of H. caprifoliatum extracts on phloroglucinol derivatives induce antidepressant-like cerebral extracellular monoamines levels, in order to activity remain unclear. Nevertheless, recently reported characterize the monoaminergic system responsible for studies have suggested that the phloroglucinol enriched the antidepressant-like effect of H. caprifoliatum. extract from H. perforatum does not act as an detergent- like substance on the membrane, since it did not affect synaptosomal membrane microviscosity up to 30 mM Acknowledgements (Gobbi et al., 1999), but through the inhibitory effect on vesicular storage by an interference with the driving This work was supported by grants from the CNPq force of the vesicular uptake (Roz et al., 2002). This (Brazil), the Centre National de la Recherche Scientifi- prompted us to investigate the effects of HC1 on que (CNRS, France) and the CAPES-COFECUB 3 synaptosomal preparations pre-loaded with [ H]-DA, International Agreement (BrazileFrance exchange pro- 2C in a Ca free modified KrebseRinger medium. We gram CAPES/COFECUB 418/03). demonstrated that exposure of the pre-loaded synapto- The authors are grateful to CNPq (Brazil), for the somes to HC1 induced a small dose-dependent increase fellowship to Stela M. K. Rates and Ana Paula Heckler; 3 in the efflux of [ H]-DA. Moreover, the HC1 concen- to CAPES, for the fellowship to Alice Fialho Viana; to 3 tration needed to induce the efflux of [ H]-DA was Sergio Bordignon, Ph.D., for collecting and identifying higher than that needed to inhibit the DA uptake. the plant material, as well as to Carolina No¨r, Michele Similar difference in potencies was previously observed Kliemann and Gustavo Provensi for technical assistance. by Gobbi et al. (1999) in the effects of the hydro- The authors also thank Richard Medeiros, Rouen methanolic extract of H. perforatum and hyperforin on University Hospital Medical Editor for his valuable 3 synaptosomal [ H]-DA uptake and release. In this study, advice in editing the manuscript. the concentration of hydromethanolic extract of H. perforatum (4000 ng/ml) or hyperforin (440 ng/ml) 3 necessary to inhibit 50% of [ H]-DA into rat striatal References synaptosomes induced, respectively, only 10.3 G 1.4% and 13.9 G 0.6% release from synaptosomes pre-loaded Barghon, R., Protais, P., Colboc, O., Costentin, J., 1981. Hypokinesia 3 with [ H]-DA. Furthermore, using in vivo microdialysis in mice and catalepsy in rats elicited by morphine associated with A. Viana et al. / Neuropharmacology 49 (2005) 1042e1052 1051

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Roz, N., Rehavi, M., 2003. Hyperforin inhibits vesicular uptake of hyperactivity. Pharmacology, Biochemistry, and Behavior 54, monoamines by dissipating pH gradient across synaptic vesicle 235e239. membrane. Life Sciences 73, 461e470. Verotta, L., Appendino, G., Jakupovic, J., Bombardelli, E., 2000. Sanchez-Mateo, C.C., Prado, B., Rabanal, R.M., 2002. Antidepres- Hyperforin analogues from St. John’s wort (Hypericum perfora- sant effects of the methanol extracts of several Hypericum species tum). Journal of Natural Products 63, 412e415. from the Canary Islands. Journal of Ethnopharmacology 79, Wonnemann, M., Singer, A., Mu¨ller, W.E., 2000. Inhibition of 3 3 119e127. sinaptossomal uptake of H-L-glutamate and H-GABA by hyper- Schmitt, A.C., Ravazzolo, A.P., Von Poser, G.L., 2001. Investigation forin, a major constituent of St. John’s wort: the role of amiloride of some Hypericum species native to Southern of Brazil for sensitive sodium conductive pathways. Neuropsychopharmacology antiviral activity. Journal of Ethnopharmacology 77, 239e245. 23, 188e197. Singer, M.S., Wonnemann, M., Mu¨ller, W.E., 1999. Hyperforin, Wu, Q.L., Wang, S.P., Du, L.J., Yang, J.S., Xiao, P.G., 1998. a major antidepressant constituent of St John’s wort, inhibits Chromone glycosides and flavonoids from Hypericum japonicum. serotonin uptake by elevating free intracellular NaC1. Phytochemistry 49, 1417e1420. Journal of Pharmacology and Experimental Therapeutics 290, Yoshitake, T., Lizuka, R., Yoshitake, S., Weikop, P., Mu¨ller, W.E., 1363e1368. O¨gren, S.O., Kehr, J., 2004. Hypericum perforatum L. (St John’s Suzuki, O., Katsumata, Y., Oya, M., Bladt, S., Wagner, H., 1984. wort) preferentially increases extracellular dopamine levels in the Inhibition of monoamine oxidase by hypericin. Planta Medica 50, rat prefrontal cortex. British Journal of Pharmacology 142, 272e274. 414e418. Vaugeois, J.M., Pouhe´, D., Zuccaro, F., Costentin, J., 1996. Indirect Yu, P.H., 2000. Effect of Hypericum perforatum extract on serotonin dopamine agonists effects on despair test, dissociation from turnover in mouse brain. Pharmacopsychiatry 33, 60e65. doi: 10.1111/j.1472-8206.2006.00440.x

REVIEW Hypericum caprifoliatum (Guttiferae) Cham. ARTICLE & Schltdl.: a species native to South Brazil with antidepressant-like activity

Alice F. Vianaa,b, Jean-Claude do Regob, Leonardo Munaria, Nathalie Dourmapb, Ana Paula Hecklera, Teresa Dalla Costaa, Gilsane L. von Posera, Jean Costentinb, Stela M.K. Ratesa* aPrograma de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul. Av. Ipiranga, 2752 Porto Alegre, RS, Brazil bUnite´ de Neuropsychopharmacologie Expe´rimentale F.R.E 2735 C.N.R.S., I.F.R.M.P. no. 23, Faculte´ de Me´decine & Pharmacie, 22 Boulevard Gambetta, 76183 Rouen Cedex, France

Keywords ABSTRACT analgesics, antidepressant, In this work, previously published and unpublished results on biological activity of depression, Hypericum caprifoliatum, a native specie to South Brazil, are presented. Lipophilic Hypericum caprifoliatum, extracts obtained from this species showed an antidepressant-like activity in mice and phloroglucinol derivatives rat forced swimming test. Results from in vivo experiments suggest an effect on the dopaminergic transmission. Besides that, in vitro experiments demonstrated that the extract and its main component (a phloroglucinol derivative) inhibit monoamine Received 31 December 2005; revised 24 February 2006; uptake in a concentration-dependent manner, more potently to dopamine, but this accepted 30 June 2006 effect is not related to direct binding at the uptake sites. It was also observed that a 3-day treatment with lipophilic extract prevents stress-induced corticosterone rise in mice frontal cortex but not in plasma. The lipophilic and methanolic H. caprifoliatum *Correspondence and reprints: extracts also demonstrated antinociceptive effect, which seems to be indirectly [email protected] mediated by the opioid system. These results indicate that H. caprifoliatum presents a promising antidepressant-like effect in rodents which seems to be related to a mechanism different from that of other classes of antidepressants.

H. perforatum extracts are used for treating mild to INTRODUCTION moderate depression. However, neither the mechanisms In the last 10 years, interest in the genus Hypericum of action nor the identity of the active constituents are (Guttiferae) has increased considerably all over the completely understood. Most authors agree that it is not world. Hypericum is a large genus of herbaceous or only one component but a combination of some of them, shrubby plants which widely occur in temperate regions such as hypericin, flavonoid derivatives and mainly of the world. This genus encompasses approximately hyperforin [4–8] that are responsible for the antidepres- 400 species [1]. Chemical investigation of the genus sant activity. With regard to the mode of action, Hypericum has led to the isolation of more than 100 lipophylic extracts and some components (e.g. hyperfo- compounds, such as flavonoids, xanthones and phloro- rin) have been shown to inhibit the uptake of glucinol derivatives [2]. Among the last type of com- noradrelanine (NA), serotonin (5-HT), dopamine (DA), pounds, some are related to the well-known hyperforin gamma-aminobutyric acid (GABA) and glutamate by a isolated from Hypericum perforatum L. while others mechanism not directly related to binding at the uptake possess a phloroglucinol unit conjugated with a filicinic sites [9–11]. Nowadays other approaches attempting to acid moiety [2,3]. explain the pharmacological properties of H. perforatum From the clinical point of view H. perforatum is the have shown that hyperforin alters sodium conductive main species. In Europe, in the USA and South America pathways [12], affects the physico-chemical properties of

ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 20 (2006) 507–514 507 508 A. F. Viana et al. neuronal membranes [13,14] and acts on hypothal- HYPERICUM CAPRIFOLIATUM amic–pituitary–adrenal (HPA) axis [15–17]. As the chemical composition and the pharmacologi- Botanic cal profile of Hypericum species are comparable, the Hypericum caprifoliatum is a subshrub 0.4–1 m tall found study of the genus Hypericum could be helpful in in forest clearings, pastures and roadsides. It has searching for alternative sources of antidepressant characteristic orange-yellow to orange pentamer small molecules and to identify drugs with innovative mech- flowers with 9–15 mm diameter stellate; buds ovoid, anisms of action. acute and sub-acute. Leaves are ovate-deltoid to oblong Brazilian Hypericum species occur predominantly at (10–22 · 7–10 mm) margin recurved to revolute, per- southern regions, where 20 of them have been identified. foliate, spreading, with sparse pale but not dark glands. Species growing in Rio Grande do Sul, the southernmost The slenderer stems, strongly connate leaves, and state in Brazil, belong to the sections Brathys and smaller sepals distinguish H. caprifoliatum from its closest Trigynobrathys, the latter with a greater number of relative H. teretiusculum, which has a more northern native species [18]. distribution [18]. In the course of our ongoing project aiming to detect bioactive molecules in southern Brazilian species of the Chemistry genus Hypericum, we selected the most widespread ones All studies were carried out with flowering aerial parts to begin the screening of their chemical composition and collected between August and December, 2001, in the pharmacological properties. Until now eight species have region of Viama˜o, in the state of Rio Grande do Sul, been studied: H. brasiliense Choisy, H. caprifoliatum Brazil. The voucher specimens were deposited at the Cham. & Schltdl., H. carinatum Griseb., H. connatum herbarium of the Federal University of Rio Grande do Sul Lam., H. cordatum (Vell.) N. Robson subsp. kleinii N. (ICN-Bordignon 1496). Robson, H. myrianthum Cham. & Schltdl., H. polyanthe- The presence of tannins and flavonoids in H. caprifo- mum Klotzsch ex Reichardt and H. ternum A. St. Hil. As liatum total methanolic extract was determined by using far as we know, none of the species is used in folk standard phytochemical methods to characterize the medicine for antidepressant purposes. Hypericum brasi- most usual plant secondary metabolites [23]. The tannin liensis and H. connatum are popularly used for relief of fraction (6.1%) includes triflavonoids and heptaflavo- disorders such as angina, cramps and ora-pharyngeal noids (three to seven condensed monomers, formed by inflammations, which could be associated with the flavan-3-ol or flavan-3,4-diol units). The non-tannin analgesic properties of these plants [19]. To the best of fraction contains phenolic compounds (monomeric fla- our knowledge, only H. brasiliense and H. cordatum have vonoid compounds and hydroxycinnamic acid deriva- been previously investigated for antidepressant activity. tives), carbohydrates (glucose, fructose, saccharose and Rats’ immobility time in forced swimming test (FST) was hydrocolloidal gums) and nitrogen compounds (such as not reduced by hydroalcoholic extracts of both species amino and imino acids). The main flavonoids are [20]; conversely, H. brasiliense extract inhibited mono- hyperoside and other glycosides of quercetin. This was amine oxidases [21,22]. expected as flavonoids are abundant in Hypericum Regarding chemical composition, these species are species [25], being quercitrin, isoquercitrin, hyperoside rich in flavonoids [23] and dimeric phloroglucinols and rutin (all derived from quercetin) the most com- conjugated with a filicinic acid moiety [24]. These monly reported glycosides [22]. phloroglucinol derivatives are proposed as chemotaxo- Hypericum species have a strong tendency to accu- nomic markers for the southern Brazilian species [25]. mulate phenolic compounds with the phloroglucinol None of the studied species presented hypericin [24]. substitution pattern. Recently two dimeric structures In this review, we brought together both published consisting of filicinic acid and a phloroglucinol moiety and unpublished data pertaining to H. caprifoliatum. This were isolated from H. caprifoliatum: hyperbrasilol [25] species was evaluated concerning its chemistry, toxicity, and a tautomeric mixture still unresolved, termed HC1 general activity on central nervous system and effects on [26]. HC1 corresponds to 8% of a purified cyclohexane animal models of anxiety, nociception and depression. extract and seems to be the active fraction on central The most promising results were found on antidepres- nervous system [26]. This type of compound has already sant-like activity which prompted us to carry out a been found in other species belonging to the sec- deeper investigation of this subject. tions Trigynobrathys – H. brasiliense [21,22], H. carin-

ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 20 (2006) 507–514 H. caprifoliatum antidepressant-like activity 509 atum, H. connatum, H. myrianthum, H. polyanthemum (piloerection, hypothermia, apnea and prostration) and [25], H. uliginosum [27], H. japonicum [28] – and Brathys – some of them died between 24 and 48 h after the last H. drummondii [29,30]. treatment. During autopsy we verified the presence of Although Hypericum species are not characteristically ascitic liquid in the abdomen. Histological and biochemi- aromatic, usually presenting no more than 1% of essential cal analysis revealed superficial liver necrosis and low oil, the presence of these compounds was also assessed. levels of albumin. These findings suggest that the death The Brazilian species, H. caprifoliatum, H. polyanthemum, was caused by circulatory collapse, i.e. in the attempt to H. myrianthum, H. carinatum, H. connatum and H. ternum dilute the extract there was a permeation of liquid into afforded essential oils in the range of 0.1–0.5%. In these the peritoneum. The lethal dose 50% (LD50) determined species, the sesquiterpene are present in higher concen- in mice by intraperitoneal route was 197 mg/kg. No tration than monoterpenes and all of them contain signs of toxicity were found when rats or mice were alkanes in the volatile fraction [24]. treated orally (50–2000 mg/kg) with petroleum ether To carry out the psychopharmacological study, sev- and cyclohexane extracts. eral extracts were obtained by continuous extraction For the assessment of cyclohexane extract (ECH) non- with solvents of increasing polarity [petroleum ether, specific effects on the central nervous system, locomotor chloroform and methanol (MET)] or by maceration with activity, motor coordination, pentobarbital sleeping time cyclohexane. Details of the extraction and purification and hypothermia were evaluated in mice treated with methods were presented elsewhere [26]. doses ranging from 90 to 360 mg/kg p.o. Treatment with ECH 90–360 mg/kg reduced locomotor activity [26,34] Pharmacology but did not interfere with motor coordination, demon- At the beginning of our project we tested the total strating that ECH neither presents a gross neurotoxicity methanolic extracts of aerial parts of several Hypericum nor affects the neuromuscular junction. The extract species in the FST which predicts antidepressant activity increased the sleeping time (saline ¼ 31.6 ± 7.8 min; [31,32]. Only H. caprifoliatum extract was active. There- ECH 90 mg/kg ¼ 77 ± 16 min) of mice treated with fore, we assayed petroleum ether, chloroform and MET pentobarbital but not the sleep latency (saline ¼ extracts in the FST, hot plate test and for monoamino 4.4 ± 0.2 min; ECH 90 mg/kg ¼ 9 ± 3.4 min). ECH oxidase inhibitory (MAOI) activity [26,31,32]. 90 mg/kg caused a significant hypothermia by itself and The most promising extract in the FST was the an enhanced apomorphine-induced (16 mg/kg, s.c.) petroleum ether which is rich in phloroglucinols. How- hypothermia [26]. These results suggest that ECH has a ever, in subsequent experiments it showed a significant depressor effect on the central nervous system or affects toxicity, thus another liphofilic extract was prepared by the metabolism of depressor substances. This latter maceration with cyclohexane. This cyclohexane extract assumption is based on studies that verified an interaction (ECH) was investigated specially toward the antidepres- of H. perforatum extracts and isolated substances with sant activity. In addition, ECH was purified yielding an metabolizing systems such as cytochrome P-450 enzyme extract free of chlorophyll and waxes termed HCP. system and pregnane X receptor [33,34]. The evaluation In vivo experiments were carried out with MET and of H. caprifoliatum action on cytochrome P-450 system is cyclohexane extracts. In vitro studies were performed under way. with HCP and with a phloroglucinols tautomeric mixture HC1, the main constituent of ECH and HCP. Anxiety and seizures All experiments were approved by CONEP, Brazil The ECH was tested in the models of anxiety such as (National Commission of Research Ethics) and performed black and white compartments and plus maze as well as according to guidelines of The National Research Ethical pentylenetetrazole induced seizures. All tests were per- Committee (published by National Heath Council – MS, formed with acute treatments with doses ranging from 1998), which are in compliance with the European 90 to 360 mg/kg p.o. The extract showed neither Communities Council Directive of 24 November 1986 anxiolytic nor anticonvulsive effects (Tables I and II). (86/609/EEC). Nociception Toxicity and general activity Methanol (MET) and the cyclohexane (ECH) extracts Rats treated with petroleum ether extract by intraperi- (90 mg/kg, i.p. and p.o.) presented an antinociceptive toneal route (270 mg/kg/day) showed signs of toxicity effect in the hot plate test [35]. The pre-treatment with

ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 20 (2006) 507–514 510 A. F. Viana et al.

Table I Effect of Hypericum caprifoliatum Plus maze Dark/light compartments cyclohexane extract (ECH) on plus maze Time in the open arm (s) Time in the light compartment (s) and dark/light compartments scores. Treatment per os (dose) Rata Miceb Micec

NaCl 0.9% (n ¼ 10) 142 ± 50 23 ± 15 131 ± 23 Diazepam (2 mg/kg; n ¼ 12) 213 ± 53* 84 ± 19*** 150 ± 25*** ECH (90 mg/kg; n ¼ 10) 171 ± 79 26 ± 14 NT ECH (180 mg/kg; n ¼ 10) NT 28 ± 14 NT ECH (360 mg/kg; n ¼ 10) NT 29 ± 7 103 ± 34

Values expressed as mean ± SD. NT, dose not tested. a One way ANOVA F2,27 ¼ 3.4 followed by multiple comparison test. Student–Newman–Keuls, *P < 0.05. b One way ANOVA F4,51 ¼ 36.7 followed by multiple comparison test. Student–Newman–Keuls, ***P < 0.001. c One way ANOVA F2,29 ¼ 11.2 followed by multiple comparison test. Student–Newman–Keuls, ***P < 0.001.

Table II Effect of Hypericum caprifoliatum Convulsion Number of Number of cyclohexane extract (ECH) on pentyl- Treatment per os latency (s)a convulsionsb deathsc enetetrazole (80 mg/kg, i.p.)-induced Diazepam (2 mg/kg; n ¼ 10) – 0 0 seizures parameters in mice. NaCl 0.9% (n ¼ 10) 62 (54–74) 11.5 ± 5 3 ECH (90 mg/kg; n ¼ 10) 57 (53–59) 18.33 ± 8 5

a b Values expressed as: median (inter-quartiles intervals), ANOVA on ranks H ¼ 4.2, mean ± SD, one-way c ANOVA F1,19 ¼ 1.2; absolute number. naloxone (2.5 mg/kg, s.c.) abolished the effect of the ECH opioid receptors is not directly related to HC1, but we i.p. and p.o., indicating that it may be mediated by the cannot rule out the possibility that HC1 metabolites, that opioid system. Conversely, antinociception produced by are not formed in vitro, might be responsible for the MET given by i.p. route was only partially prevented analgesic activity. Furthermore, we can reason that the by naloxone and the antinociceptive activity per os antinociceptive effect of ECH is indirect by acting in other (p.o.) was not modified. Furthermore, ECH significantly neurotransmitter systems (e.g. dopaminergic system) reduced the number of abdominal writhing induced by rather than modulated by the opioid functions. acetic acid while MET showed only a mild and not significant effect. Depression As mentioned in the section ‘Chemistry’, the metha- Some of us have reported the effect of H. caprifoliatum in nolic extract is rich in flavonoids while the main the rat FST [31]. This study revealed that among the compound of the cyclohexane extract (ECH) is a phlo- investigated extracts (petroleum ether, chloroform and roglocinol derivative tautomeric mixture (HC1). There- MET), the only one displaying an antidepressant-like fore we tested the fractions from each extract, enriched effect was the petroleum ether (270 mg/kg/day, i.p.). ) in flavonoids or HC1, in the hot plate test. Unexpectedly, This extract (1.5 · 10 2 mg/mL) was submitted to none of the fractions had antinociceptive effect. In in vitro monoamino oxidase (MAO) inhibition assays addition, we verified that the phloroglucinol mixture and revealed an inhibition of no more than 25% on both ) ) HC1 (10 6–3 · 10 11 g/mL) did not affect the binding of MAO-A and MAO-B. This magnitude of effect was 3 [ H]-naloxone (1 nM) to the rats’ total brain membrane. considered too low to explain the antidepressant-like The summation of results indicates that the antinoc- effect observed in the FST [32]. iceptive effect of ECH and MET may be attributed to a The antidepressant-like effect of H. caprifoliatum synergistic effect of substances rather than the effect of a petroleum ether extract is retained by other lipophilic single one. MET seems to have at least two groups of and purified extract rich in phloroglucinols (ECH). substances with distinct pharmacokinetic profiles and We observed that ECH shows a dose-dependent (90– mechanisms of action [35]. The effect of ECH on the 360 mg/kg, p.o.) effect when evaluated on rat and mice

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FST [26]. Furthermore, the anti-immobility effect on FST effects on dopamine uptake and release, acute treatment was not due to a nonspecific behavioral stimulation as with ECH (270 mg/kg, p.o.) did not increase dopamine, ECH 90 mg/kg p.o. also reduces mice locomotor activity homovanilic acid (HVA) and 3,4-dihydroxyphenylacetic as shown in general activity studies. This observation acid (DOPAC) extracellular levels in the accumbens reinforces the hypothesis that ECH has antidepressant nucleus (coordinates from bregma: A +2.2; L )1.5; D )6) properties. Given this, to be considered as a potential measured by microdialysis in awake rats, using high- antidepressant, a drug must reduce immobility in FST at pressure liquid chromatography with electrochemical doses that do not stimulate locomotion [36]. This result detection. This fact could be a consequence of low ECH or is also in accordance with studies which show that many HC1 systemic levels. The evaluation on striatum DA antidepressants tend to decrease motor activity [37,38]. levels as wells as other treatment regimens and the Aiming to study the mode of action of H. caprifoliatum pharmacokinetic plasma profile of ECH is in progress. lipophilic extracts, the second set of experiments was Moreover, contrary to all other known antidepressant performed to evaluate whether the monoaminergic drugs, the inhibition of monoamine uptake operated by system is involved in anti-immobility effect. The sum- H. caprifoliatum extracts is not due to HC1 binding or DA, mary of the results is presented next. Methodological NA or 5-HT transporters blockage at the binding sites details and full data are presented elsewhere [26]. The of [3H]-mazindol, [3H]-nisoxetine and [3H]-citalopram, effect of ECH in mice FST was prevented by a prior respectively [26]. These observations suggest that the administration of either sulpiride or SCH 23390 (D2 and mechanism of action of H. caprifoliatum extract is D1 dopamine receptor antagonist, respectively). These probably not associated with a specific binding to either results led us to further investigate ECH and HC1 one of the different monoamine transporters, but could regarding their action on monoaminergic system, more depend on a mechanism involved in neurotransmitters precisely on dopamine. ECH was purified as previously transport in general. described [26] furnishing HCP which has a chemical This assumption had been previously reported for profile comparable to ECH: both extracts present HC1 other species of Hypericum, such as H. perforatum [39–42] as the main constituent. HCP and HC1 inhibited, in and H. triquetrifolium [44]. In these reports, several in a concentration-dependent and monophasic manner, vivo and in vitro animal studies have demonstrated that [3H]-DA, [3H]-NA and [3H]-5HT synaptosomal uptakes the antidepressant properties and the inhibitory effects of (Table III). This inhibition was stronger for DA than NA the extracts from these species on the neuronal uptake and 5-HT confirming the dopaminergic feature of H. cap- of the monoamines were due to the lipophilic phloro- rifoliatum antidepressant-like effect. It appears that these glucinol derivative, hyperforin [39,41,43–45], which is effects are related to the phloroglucinol derivative present considered the main active substance of H. perforatum in ECH as it inhibited [3H]-DA synaptosomal uptake with and H. triquetrifolium. Comparing our results with a potency equivalent to ECH although HC1 constitutes H. perforatum data, we can point out that although 3 only 8% of ECH. When tested at concentrations corres- ECH and HC1 IC50 for [ H]-DA uptake inhibition (98 and 3 ponding to its IC50 on [ H]-DA uptake, HC1 did not 85 ng/mL, respectively) are about twice as high as that 3 induce a significant [ H]-DA release, while at a higher of hyperforin- (55 ng/mL) and hyperforin-enriched CO2 concentration (200 ng/mL) it significantly enhanced (by extract (56 ng/mL) [44], H. caprifoliatum appears to have 12%) the synaptosomal DA release [26]. Despite the the same range of potency of other Hypericum species in

Table III Comparison of the inhibition of Uptake of monoamine uptake by HCP and HC1. [3H]-DA [3H]-5HT [3H]-NA

IC50 (ng/mL) n Hill IC50 (ng/mL) n Hill IC50 (ng/mL) n Hill

HCP 98 ± 18 1.0 ± 0.1 520 ± 32 0.8 ± 0.1 971 ± 98 0.7 ± 0.1 HC1 85 ± 19 1.2 ± 0.2 599 ± 60 0.7 ± 0.1 206 ± 24 0.7 ± 0.1

Data were calculated using the equation of the curve-fitting programs MICROCAL ORIGIN (Microcal Software Inc.,

Northampton, MA, USA). IC50 and n Hill values represent the mean ± SEM of four independent experiments, carried out in duplicate. Data from our earlier results [26].

ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 20 (2006) 507–514 512 A. F. Viana et al. the FST. In order to obtain an effect similar to imipramine [11,26,39,43]. Besides that, the fact that Hypericum 30 mg/kg/day in the rat FST, a dose of 270 mg/kg/day phloroglocinol derivatives do not have nitrogen atoms in of ECH was required, while the effective doses of their structures is remarkably against the structure– alcoholic extracts from other species ranged from 300 activity relationship studies for monoamines receptors to 3000 mg/kg/day in rats [41,45]. In the mice FST the and transporters [50,51] which may account for its effective doses of other Hypericum extracts are between particular mechanism of action. Consequently, the 250 and 1000 mg/kg [46,47], while for ECH the lowest research on substances from this genus could result in effective dose is 270 mg/kg. antidepressant molecules with innovative mechanisms of Nevertheless, despite all studies on the antidepressant- action and also give insights into the pathophysiology of like activity of the genus Hypericum, the molecular depression. mechanisms by which phloroglucinol derivatives, such as HC1, induce antidepressant-like activity remain ACKNOWLEDGEMENTS unclear. Recently, a second line of investigation along- side the monoaminergic approach has been focusing on This work was supported by grants from the CNPq the action of antidepressants in the HPA axis [48]. (Brazil), the Centre National de la Recherche Scientifique We investigated the effect of ECH (360 mg/kg, p.o.) on (CNRS, France), the CAPES-COFECUB International serum and brain corticosterone levels of mice, stressed or Agreement (Brazil-France exchange program CAPES/ not by FST [A. F. Viana, unpublished data]. In brief, COFECUB 418/03) and International Foundation for cortical but not serum corticosterone FST-induced rise Science (IFS, agreement F/3425-1). The authors was diminished by repeated (3 days) ECH treatment; thank Ms Andresa Betti for revising the English of the acute (1 day) treatment did not reduce FST-augmented manuscript. corticosterone; basal corticosterone levels (non-stressed animals) were not reduced by either treatment regimes. REFERENCES In parallel, we demonstrated that repeated administra- tion of imipramine and bupropion reduces both cortical 1 Robson N.K.B. Studies in the genus Hypericum L. (Guttiferae) 2. and serum corticosterone levels. 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(1988) 154 185–190. gamma-pyrone and xanthones with monoamine oxidase 38 Bourin M. Is it possible to predict the activity of a new inhibitory activity from Hypericum brasiliense. Phytochemistry antidepressant in animals with simple psychopharmacological (1994) 36 1381–1385. test? Fundam. Clin. Pharmacol. (1990) 4 49–64. 22 Rocha L., Marston A., Potterat O., Kaplan M.A., Stoeckli-Evans 39 Chatterjee S.S., No¨ldner M., Koch E., Erdelmeier C. Antidepres- H., Hostettmann K. Antibacterial phloroglucinols and flavo- sant activity of Hypericum perforatum and hyperforin, the noids from Hypericum brasiliense. Phytochemistry (1995) 40 neglected possibility. Pharmacopsychiatry (1998) 31 7–15. 1447–1452. 40 Mu¨ller W.E., Rolli M., Schafer C., Hafner U. Effects of Hypericum 23 Dall’Agnol R., Ferraz A., Bernardi A.P. et al. Antimicrobial extract (LI 160) in biochemical models of antidepressant activity of some Hypericum species. Phytomedicine (2003) 10 activity. 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44 Chatterjee S.S., Bhattacharya S.K., Wonnemann M., Singer A., two of its constituents on neuroendocrine responses in the rat. Mu¨ller W.E. Hyperforin as a possible antidepressant component J. Psychopharmacol. (2000) 14 360–363. of Hypericum extracts. Life Sci. (1998) 63 499–510. 49 Juruena M.F., Cleare A.S., Pariante C.M. The hypothalamic- 45 Jensen A.G., Hansen S.H., Nielsen E.O. Adhyperforin as pituitary adrenal axis, glucocorticoid receptor functions and contributor to the effect of Hypericum perforatum L. in bio- relevance to depression. Rev. Bres. Psiquiatr. (2004) 26 189– chemical models of antidepressant activity. Life Sci. (2001) 68 201. 1593–1605. 50 Claramunt R., Manaut F. Para´metros o factores descriptores de 46 O¨ ztu¨rk Y., Aydi S., Beis R., Baser K.H.C., Berberoglu H. Effects of las propriedads fisicoquı´micas de los compuestos orga´nicos y Hypericum perforatum L. and Hypericum calycinum L. extracts on relaciones cuantitativas estructura-activida (QSAR) en el disen˜o the central nervous system in mice. Phytomedicine (1996) 3 de fa´rmacos, in: Avendan˜o C. (Ed.), Introduccion a la quı´mica 139–146. farmace´utica, MacGraw-Hill – Interamericana, Madrid, Espan˜a, 47 Sanchez-Mateo C.C., Prado B., Rabanal R.M. Antidepressant 1996, pp. 73–100. effects of the methanol extracts of several Hypericum species from 51 Appell M., Berfield J.L., Wang L.C., Dunn W.J., III, Chen N., the Canary Islands. J. Ethnopharmacol. (2002) 79 119–127. Reith M.E.A. Structure–activity relationships for substrate 48 Franklin M., Chi J.D., Mannel M., Cowen P.J. Acute effects of recognition by the human dopamine transporter. Biochem. LI 160 (extract of Hypericum perforatum, St John’s wort) and Pharmacol. (2004) 67 293–302.

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CHAPTER 2: Effects of Hypericum caprifoliatum Cham. & Schltdt. (Guttiferae) cyclohexane extract on serum and brain corticosterone levels

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2.1. INTRODUCTION

In this chapter, we continued the investigation of Hypericum caprifoliatum cyclohexane extract (HCP) mechanism of action. Now we carried out a less conventional approach, by studying HCP influence on the hypothalamic-pituitary-adrenal axis (HPA). As highlighted in the review section, mood disorders often coincide with abnormal levels of corticosteroids. Likewise, it has long been observed that patients with Cushing’s syndrome, hyperactivity of the HPA axis that can be induced pharmacologically or by pituitary or adrenal tumors, have high comorbity of psychiatric disturbances, including depression (Starkman et al., 1981; Becker et al., 1983). During the past decade, several research groups formulated a hypothesis relating aberrant stress hormone deregulation to causality of depression and submitted that antidepressants may act through normalization of these HPA changes (Gold et al., 1995; Holsboer and Barden 1996; Nemmeroff, 1996). Therefore HPA axis may be an important target to develop antidepressant action (Barden et al., 1995; Gold et al., 1995; Holsboer and Barden, 1996; Pariante et al., 2004).

In essence, stress underlies most animal models predictive of antidepressant acitivity. Stressful conditions such as electrical paws stimulations performed in the learned helplessness test (Foa et al., 1992; Henn et al., 1993; Petty et al., 1997), chronic mild stress (Willner et al., 1987, 1997, 2005; Moreau et al., 1992, 1994; Auriacombe et al., 1997), forced swimming test (Porsolt et al., 1978), are known to induce in rodents depressive like symptoms that are reversed by antidepressants. Furthermore, models that use animals selectively bred for presenting neurochemical and behavior alterations parallel to those found in human depressed patients, present alterations in the HPA axis, that are corrected after antidepressant treatment (El Yacoubi et al., 2003; Morley-Fletcher et al., 2004). Accordingly, it has been observed that the hyperactivity of the HPA axis in animals and depressed patients was corrected by antidepressant drugs such as imipramine, moclobemide, fluoxetine (Raap and van de Kar, 1999; Holsboer, 2000; Pariante and Miller, 2001).

The antidepressant plant H. perforatum have demonstrated interesting effects on HPA axis. Franklin and colleagues showed that the acute treatment of rats with H. perforatum causes an elevation of plasma corticosterone (Franklin et al., 2000), whereas 15 days

106 Confidencial 23/3/2007 treatment reduces brain levels of cortisol and corticosterone (Franklin et al., 2004). The treatment of rats with flavonoids isolated from H. perforatum for two weeks down-regulated circulating plasma levels of ACTH and corticosterone (Butterweck et al., 2004). The salivary concentration of cortisol under cortisol infusion was decreased in patients treated with H. perforatum when compared to placebo (Murck et al., 2004). Long-term pre-treatment of rats with H. perforatum extracts and hypericin significantly decreased levels of corticotropin- releasing hormone (CRH) mRNA in the hypothalamic paraventricular nucleus and reduced the stress-induced increases in gene transcription of proopiomelanocortin (POMC) in the anterior pituitary, glutamic acid decarboxylase (GAD 65/67) in the bed nucleus of the stria terminalis (BST) and cyclic AMP response element binding protein (CREB) in the hippocampus (Butterweck et al., 2001). Conflicting results were reported by Webber et al. (2006) for LI160 H. perforatum extract, both acute and 2 weeks treatments increased brain and plasma corticosterone levels in non-stressed mice.

Thus, the results obtained for H. perforatum, together with the importance of HPA axis for most of the comercial available antidepressant drugs, prompted us to study the effects of acute and repeated administration of H. caprifoliatum, compared to that of imipramine and bupropion, on serum and brain frontal cortex corticosterone levels in mice submitted or not to forced swimming test (FST).

2.2. BACKGROUND

The successful behavioral adaptation to stress modulated by steroid effects on higher brain functions involves negative feedback action via the paraventricular nucleus (PVN) of hypothalamus and neural inhibitory circuits that overcome the excitatory extra-hypothalamic influences on HPA axis. If stress control fails, an imbalance between stress stimuli and negative feedback develops at the different levels of the HPA axis such as: altered expression of corticotrophin releasing hormone (CRH) and vasopressin (VP) at the PVN of hypothalamus, and adrenocorticotropic hormone (ACTH) at pituitary. The extention of the alterations is dependent on the nature and context of the stress. How the imbalance between

107 Confidencial 23/3/2007 excitatory and inhibitory signals develops is not known. Whatever the cause, in the end, after the adaptation of HPA axis, the imbalance leads to altered sensitivity to corticoids hormones at the adrenal level, down-regulation of hipocamppal glucocorticoid receptors (GR), and alterations on the negative feedback mechanisms resulting in incresead levels of hormones and peptides (de Kloet et al., 1998; Sapolsky, 2001; Reul and Holsboer, 2002). These modifications are observed in patients with mood disorders, being documented the hypersecretion of CRH, ACTH and cortisol, exaggerated cortisol response to ACTH as well as blunted ACTH response to a CRH and a relative resistance to the dexamethasone suppressive effect on cortisol (Sachar et al., 1976; Gold et al., 1996; Holsboer and Barden, 1996). Postmortem samples revealed enlargement of pituitary and adrenal glands, increased CRH messenger RNA (mRNA) and downregulation of CRH receptors in the frontal cortex of victims of suicide (Nemeroff 1996).

Cortisol and corticosterone actions in the brain are mediated by glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs). GRs occur everywhere in the brain but are most abundant in hypothalamic CRH neurons and pituitary corticotropes. Most MRs in the brain is located in the hippocampus where they may be co-expressed with GR by some neurons (de Kloet et al., 1998). Hippocampal MRs bind the glucocorticoid cortisol and corticosterone with approximately tenfold higher affinity than GRs. This “nonselectivity” of brain MRs is determined by the fact that unlike in peripheral cells, a different isoform of the steroid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) is present in the hippocampus, which does not effectively exclude corticosterone or cortisol from MR-targets in this structure (Seckl, 1997). Since hippocampal MRs have about tenfold higher affinity for corticosterone, these receptors are already almost completely occupied at basal levels of corticosteroid secretion. On the other hand, hippocampal GRs are only occupied when corticosteroid levels increase under stress conditions or at the peak of the circadian rhythm of corticosteroid secretion (Holsboer, 2000). The coexistence of MRs activated at low corticosteroid concentrations, and of GRs activated only at high concentrations, allows the brain to differentially respond to the wide range of concentrations over which corticosteroids are secreted. At low concentrations, cortisol/corticosterone maintains neuronal excitability

108 Confidencial 23/3/2007 which is a predominantly MR governed effect, whereas at higher hormone concentrations this is opposed by increasing GR activation (Joëls and de Kloet 1992; Reul and Holsboer, 2002).

Chronic stress increases glucocorticoid levels and decreases BDNF (brain derived neurotrophic factor) and thereby causes atrophy or, in severe cases, death of hipocamppus CA3 neurons. These effects of stress could contribute to the observations of decreased volume and function of hippocampus in depressed patients. In addition, subtle damage of neurons by prior exposure to a neuronal insult could explain the selective vulnerability of certain individuals to become depressed when exposed to stress. Genetic factors could also contribute to such selective vulnerability. Studies with depressed patients, animals and cellular models have demonstrated that antidepressants increase expression and function of GR, and promote its nuclear translocation, these effects being associated with normalization of GR-mediated negative feedback by endogenous glucocorticoids (Pariante and Miller, 2001). Chronic antidepressant treatment increases the expression of BDNF and TrkB, and prevents the down-regulation of BDNF in response to stress. This could reverse the atrophy of hippocampal neurons, as well as protect these neurons from further damage. Up-regulation of BDNF and TrkB occurs via increased 5-HT and NE neurotransmission and up-regulation of the cAMP–CREB cascade (Duman et al., 1998; Duman and Monteggia, 2006). This cascade of events is depicted in the figure 2.1.

Therefore, antidepressant’s effect on monoaminergic systems may lead to the normalization of HPA axis through limbic system connections. Serotonin has been implicated in the stress-related regulation of the HPA axis. The dorsal raphe nucleus (DRN) and median raphe nucleus (MRN) innervate both limbic and HPA axis structures (Cooper, 1996). Serotonergic fibers from the DRN and the MRN innervate CRH producing cell bodies in the PVN of hypothalamus and there are serotonergic neurons located entirely in the hypothalamus. There is evidence that central serotonergic systems exert a positive control on the HPA axis and that reciprocally, glucocorticoids and catecholamines mediate stress- induced alterations in the central serotonergic systems (Tafet and Bernardini, 2003). Adrenaline and noradrenaline belong to the other main system of adaptative response to stress, the sympatho-adrenergic-noradrenergic (SAN) axis. The SAN and HPA axis produce

109 Confidencial 23/3/2007 mutual positive regulation; the activation of one involves the activation of the other and vice- versa (Abercrombie and Zigmond, 1995). The locus coeruleus (LC) projects noradrenergic fibers to various structures, including amygdala, hippocampus and PVN of hypothalamus (Charney et al., 1995). In association with the LC, the lateral hypothalamus mediates the activation of the sympathetic component of the autonomic nervous system (ANS). Dopaminergic system is also implicated in stress-related regulation of HPA axis and depression (Nestler and Carlezon, 2006). The mesolimbic and mesocortical dopaminergic projections are involved in adaptational process; the first one processes and reinforces rewarding and motivational stimuli; and the second one is involved in cognitive functions such as evaluation potentially stressful situations (Le Moal, 1995). The role of hypothalamus in reward mechanisms is well recognized by means of self stimulation experiments (Nestler and Carlezon, 2006). Acute stress increases mesolimbic dopamine (DA) release (Imperato et al., 1993; Lindley et al., 1999; Dazzi et al., 2001). There is evidence that dopamine agonists exert a positive control on the HPA axis. Activation of brain dopaminergic receptors, by pergolide and chemical analogs, leads to increased serum corticosterone concentration in rats, and this effect is reversed by dopamine antagonists (Fuller and Snoddy, 1983, 1984; Foreman et al., 1989).

Pariante et al. (2001) suggested that the interactions between antidepressants and glucocorticoids also may be mediated via the multiple drug resistance (MDR) P-glycoprotein (Pgp). The MDR-Pgp expression in endothelial cells of capillary blood vessels at the blood brain barrier (BBB) was first reported by Cordon-Cardo et al. (1989), and associated to brain protection against apolar xenobiotics and resistance to some cancer chemotherapies. Many other substances as modern histamine H-1 receptor antagonists, calcium channel blockers, antidepressants are also Pgp substrates and/or inhibitors (Pariante et al., 2001; Weiss et al., 2003; Loscher and Potschka, 2005). Nowadays it is established that some GR ligands, like cortisol and dexamethasone, are actively excreted from cells by the MDR-Pgp and other membrane transporters also belonging to the ATP-binding cassette transport family (Muller et al., 2003). Based on these findings, a relationship between Pgp activity at BBB and the regulation of the HPA system under both basal and stress conditions has been suggested. It is proposed that antidepressants could inhibit steroid transporters localized on the BBB in

110 Confidencial 23/3/2007 humans, increasing the access of cortisol to the brain and, in consequence, the glucocorticoid-mediated negative feedback on the HPA axis (Pariante and Miller, 2001; Juruena et al., 2004; Pariante et al., 2004). Other factors that control the access of natural or synthetic corticosteroids to the brain receptors as the corticosteroid-binding globulin (CBG) and the isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD 1 and 2), were not yet clearly correlated with depression (Webber et al., 2000; Poor et al., 2004; Holmes et al., 2006).

Figure 2.1. The influence of stress and antidepressant treatments on CA3 pyramidal neurons in the hippocampus (Duman et al.,1997).

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2.3. MATERIALS AND METHODS

2.3.1. Animals

Male Swiss albinos CD1 mice (IFFA-CREDO/Charles River, Saint-Germain sur L’Arbresle, France), weighing 25-30 g, were housed 20 in Makrolon cages (L : 40 cm, W : 25 cm, H : 18 cm), with free access to standard semi-synthetic laboratory diet (U.A.R., Villemoisson sur Orge, France) and tap water ad libitum. The animals were kept in a ventilated room, at a temperature of 22 ± 1°C, under a 12-h light/12-h dark cycle (light on between 7:00 a.m. and 7:00 p.m.). On the day before sacrifice, in order to become familiar with the environment, mice were placed in an experimental room with food and water available ad libitum and with the same housing conditions.

All the experiments were carried out between 8:00 a.m. 12:00 a.m. in testing rooms adjacent to the animal rooms. Animal manipulations were performed according to the European Communities Council Directive of November 24th 1986 (86:609:EEC), approved by Regional Ethical Committee for Animal Experimentation (Normandy; no. 09-04-04-11) and conducted by authorized investigators.

2.3.2. Drugs

Hypericum caprifoliatum extract (HCP): The aerial parts of H. caprifoliatum were collected in the region of Viamão in the state of Rio Grande do Sul - Brazil (August/2003). The voucher specimen was deposited in the herbarium of the Federal University of Rio Grande do Sul (ICN) (Bordignon, 1496). The dried and powdered plant material (120 g of aerial parts) was extracted with cyclohexane (plant/solvent ratio 1:10 w/v) by maceration (3x24h), followed by evaporation to dryness under reduced pressure at 45ºC yielding an extract named HCP (c.a. 5.0 g).

Bupropion HCl, imipramine HCl, corticosterone and rabbit antibodies against corticosterone were obtained from Sigma Aldrich, Saint-Quentin Fallavier (France); cyclohexane, and polysorbate 80 from Merck, Darmstadt/DE; tritiated corticosterone ([1,2,6,7(N)]3H-corticosterone), specific activity 81 Ci/mmol from Amersham, Orsay, (France).

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2.3.3. Treatments and procedures

Acute treatment: groups of 30 animals were treated by gavages with HCP (360 mg/kg), imipramine (IMI - 20 mg/kg), bupropion (BUP - 30 mg/kg) or saline (with 10% polysorbate 80). Repeated treatment: groups of 30 animals were treated once daily with the same doses as in the acute treatment, during 3 days.

In each of preceding groups half of the animals (n = 15) were submitted to a single 6 min forced swimming session (FSS), 1h after the single or the third treatment on the repeated one.

All procedures (mice isolation, forced swimming session and decapitation) were performed respectively in separated rooms.

2.3.4. Forced-swimming test

The forced-swimming test was essentially similar to that described by Porsolt et al. (1977). The apparatus consisted of two Plexiglas cylinders (20 cm height, 14 cm internal diameter) placed side by side in a Makrolon cage (38 x 24 x 18 cm), filled with water, maintained at 22 ± 1°C, to a height of 12 cm instead of 6 cm suggested by Porsolt et al. (1977). Two mice were tested simultaneously for a 6-min period inside vertical Plexiglas cylinders; an opaque screen placed between the two cylinders prevented mice from seeing each other. The total duration of immobility was measured, during a 6 minutes test period, with an automated image analysis system (Videotrack MV 45 system).

2.3.5. Corticosterone assay

Cortical and serum corticosterone levels were determined by radioimmunoassay (RIA). Serum corticosterone level was assayed as described by Le Cudennec et al. (2002). Mice submitted to FSS were sacrificed 30 min after the swimming session. Mice not submitted to FSS were sacrificed 1h after the last treatment. Trunk blood was collected and was centrifuged (13,000 g; 4°C; 15 min) after clotting. Serum was collected, centrifuged again (13,000g, 4°C, 15 min) and aliquots of 100 µl were stored at – 40°C until RIA.

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For cortical corticosterone measurements, the frontal cortex, dissected according to Glowinski and Iversen (1966), of 3 mice was weighed (approximately 125 mg) and homogenized in 1 ml phosphate buffer (pH 7.4) containing 1% bovine serum albumin (BSA) and stored at – 40°C until RIA.

Cortex homogenates (1 ml) and serum aliquots (11 µl) were extracted by mixing with 2 ml of ethyl acetate, followed by centrifugation (3000 g; 4°C; 5 min) to speed up phase separation. Aliquots of 1.5 ml of the ethyl acetate phase were evaporated to dryness. Samples and standards (39 - 10,000 pg/tube) were incubated at 4°C overnight in a final volume of 1 ml with rabbit antibodies against corticosterone and tritiated corticosterone. The separation of free and bound corticosterone was performed, at 4°C, by adding 500 µl of dextran-coated charcoal suspension. Ten minutes later, samples were centrifuged (3300 g; 4°C; 15 min) and radioactivity contained in the supernatant was measured by liquid scintillation (Tri-carb 2100TR, Packard Bioscience Co). The intra-assay and inter-assay coefficients of variation were 3.6 and 5.2% respectively.

2.6. Statistical analysis

Results are expressed as means ± S.E.M. Statistical of differences between groups was performed by using ANOVA (one or two factors) followed by Newman-Keuls multiple comparison tests. A probability levels of 0.05 or smaller was used to indicate statistical significance.

2.4. RESULTS

2.4.1. Effect of acute and repeated treatment with IMI, BUP and HCP on swim stress- induced immobility As expected (Viana et al., 2005), acute administration of IMI (20 mg/kg, per os), BUP (30 mg/kg, per os) or HCP (360 mg/kg, per os) significantly (p < 0.05 - 0.001) reduced the immobility time induced by forced swimming test, compared to saline treated mice (Figure

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2.2). When administrated daily during three days, IMI (20 mg/kg, daily), BUP (30 mg/kg, daily) or HCP (360 mg/kg, daily) also provoked a significant (p < 0.01) reduction in the immobility time (Figure 2.2). The repeated treatment effect of IMI and HCP was somewhat more important than that measured in mice treated acutely. The percentage of immobility time reduction in mice treated three days with IMI (52.7 ± 5.1%) or HCP (39.6 ± 2.5%) differs significantly (p < 0.05 – 0.01) from that of mice treated acutely (36.3 ± 2.4% and 29.2 ± 2.2% for IMI and HCP respectively). The percentage of immobility time reduction in mice treated with BUP in the acute treatment was 63.2 ± 5.1% and did not change after three days treatment 63.3 ± 6.6%.

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(A) 200

150

* ) s

( ** y t i l 100 obi

m m *** I

50

0 CONT SAL IMI BUP HCP

(B) 250

200

) s ( 150 *** y t i l *** obi

m 100

m *** I

50

0 CONT SAL IMI BUP HCP

Figure 2.2. Effect of (A) acute (1 day) or (B) repeated treatment (3 days) with imipramine (IMI 20 mg/kg), bupropion (BUP 30 mg/kg), H. caprifoliatum (HCP 360 mg/kg) or saline (SAL), CONT (naive mice) on forced swimming test. 60 minutes after the last treatments, the animals were submitted to the swimming test, and the total duration of immobility was measured during a 6-min test period. Data are presented in mean ± SEM (n = 15 mice/group). Significantly different values were detected by one way ANOVA followed by post hoc Student- Newman-Keuls test : *p < 0.05; **p < 0.01; ***p < 0.001 compared to their respective saline (SAL) group.

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2.4.2. Effect of forced swimming test on serum and cortical corticosterone levels The FST significantly (p < 0.001, Student- Newman-Keuls test) increased both serum (+400%) (Figure 2.3A, Figure 2.4A) and cortical (+600%) (Figure 2.3B, Figure 2.4B) corticosterone levels, measured 30 min later. These effects were similar whether animals were treated with saline or were naïve mice (CONT).

2.4.3. Acute effect of IMI, BUP or HCP on serum and cortical corticosterone levels Acute treatment with IMI (20 mg/kg, p.o.) or BUP (30 mg/kg, p.o.) did not have significant effect neither on serum (Figure 2.3A) nor on cortical (Figure 2.3B) corticosterone levels, both in mice submitted or not to a FST. Acute treatment with HCP (360 mg/kg, p.o.), also did not cause significant effect on FST-induced increase of serum and cortical corticosterone levels, but produced a marked (p < 0.001) increase in basal serum corticosterone level (Figure 2.3A). It is noteworthy that, although not significantly, HCP also induced a slight increase of cortical corticosterone level (Figure 2.3B).

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basal after FST (A) 60 c

e

n 50 c ) c c *** m tero

s 40

o c seru

rti 30 co c

i 100 ml 20 / g

µ (

asmat 10

Pl 0 CONT SAL IMI BUP HCP

(B) 160 c c c 140 c c

e

n 120

ro e

e) 100 t s

o

ssu 80 c i i t rt 60 co mg /

g 40 p cal ( i

rt 20

Co 0 CONT SAL IMI BUP HCP

Figure 2.3. Effect of acute treatment (1 day) with imipramine (IMI 20 mg/kg), bupropion (BUP 30 mg/kg), H. caprifoliatum (HCP 360 mg/kg) or saline (SAL) on serum (A) and cortical (B) corticosterone levels, in mice submitted or not to forced swim stress. Data are presented in mean ± SEM (n = 15 mice/group). Significantly different values were detected by two way ANOVA followed by post hoc Student-Newman-Keuls test : cp < 0.001 compared to respective no swim stressed group; ***p < 0.001 compared to respective saline (SAL) group.

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2.4.4. Repeated treatment effect of IMI, BUP or HCP on serum and cortical corticosterone levels After three days of treatment, the increase of both serum and cortical corticosterone levels elicited by FST was significantly (p < 0.01-0.001) reduced by IMI (20 mg/kg, daily) and BUP (30 mg/kg, daily), while they decreased slightly but not significantly (two way ANOVA) both serum and cortical basal corticosterone levels in mice without swim stress (Figure 2.3). It is noteworthy that, when compared with one way ANOVA, separately from the stress factor, there are a significant difference (p < 0.05) among the basal corticosterone levels of group treated with saline and IMI or BUP. The repeated treatment with HCP (360 mg/kg, daily) significantly reduced only the cortical corticosterone level (Figure 2.3B) but not serum corticosterone level (Figure 2.3A). In this treatment group, the basal serum corticosterone level in mice without stress was not markedly modified (Figure 2.3A), whereas the basal cortical corticosterone level in mice without stress was slightly, but not significantly, increased (Figure 2.3B). In percentage terms, the reductions in serum and cortical corticosterone levels- induced by FST were respectively: 58.9 and 57.2% for IMI; 36 and 46.2% for BUP; and 24 and 49.9% for HCP, relatively to respective saline treated mice.

119 Confidencial 23/3/2007 basal after FST (A)

60

e

n 50 ) ro c e c m t

s 40

o c c

i seru t r 30 c ** o

c i t 100 ml

/ 20 b a *** g m µ ( as

l 10 P

0 CONT SAL IMI BUP HCP

(B)

160 c e

n 140 c ro e e)

t 120 s o ssu c

i 100 i c

t *** rt 80 co mg c *** / *** g p cal 60 ( i rt 40 Co 20

0 CONT SAL IMI BUP HCP

Figure 2.4. Effect of repeated treatment (3 days) with imipramine (IMI 20 mg/kg daily), bupropion (BUP 30 mg/kg daily), H. caprifoliatum (HCP 360 mg/kg daily) or saline (SAL) on serum (A) and cortical (B) corticosterone levels in mice submitted or no to forced swim stress. Data are presented in mean ± SEM (n = 15 mice/group). Significantly different values were detected by two way ANOVA followed by post hoc Student-Newman-Keuls test : bp < 0.01, cp < 0.001 as compared to respective no swim stressed group; **p < 0.01, ***p < 0.001 as compared to respective saline (SAL) group.

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2.5. DISCUSSION

In this study we have confirmed that a forced swimming test (FST) is a major stressor in mice (Marek et al., 1993; Grosel et al., 1993; Mogil et al., 1996). In all experiments, cortical and serum corticosterone levels were significantly increased by forced swimming session in control animals or in mice treated with saline. This marked increase in serum and brain corticosterone levels reflects an activation of the hypothalamo-pituitary-adrenal (HPA) axis. This neuroendocrine activity induced by forced swimming was significantly reduced, in both serum and frontal cortex, by repeated treatments (3 days) with imipramine and bupropion. To the best of our knowledge, this is the first report of a prevention of a stress-induced increase in serum or cortical corticosterone levels elicited by antidepressants after a so brief treatment. Previous studies had practiced acute (Delbende et al., 1991) and chronic treatments (14 – 30 days) and measured only plasma or serum corticosterone (Holsboer, 2000; Pariante and Miller, 2001) with few reports to brain levels (Weber et al., 2006).

When administered acutely none of the antidepressants was effective in reducing FST- induced increase in serum or cortical corticosterone levels, despite of their anti-immobility effects. Thus, it appears that the reduction in FST immobility time after the acute administration of drugs is not linked to a decrease in either serum or cortical corticosterone. This is reinforced by the fact that BUP reduces serum and cortical corticosterone response to FST only when administered during three days; it reduces the immobility in the FST with the same magnitude in both acute and repeated treatment. Conversely, the reduction of FST immobility time induced by IMI and HCP is more pronounced after repeated than after acute treatment. The lack of more pronounced decrease in immobility time, in mice repeatedly treated with BUP makes uneasy the establishment of a correlation between the two effects. One may notice that, among the three tested drugs, BUP is the only to induce stimulant locomotor effect (Nielsen et al., 1986; Zarrindast and Hosseini-Nia, 1988; Martin et al., 1990; Ascher et al., 1995), which may interfere with the immobility time in the FST. Considering all these points, one cannot establish a relationship between the decrease in corticosterone response to FST and the reduction in immobility time during the FST after the third injection of tested drugs.

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Similarly to IMI and BUP, HCP had no acute effect on stressed mice corticosterone levels and was effective in reducing the immobility time in the FST in both treatment regimens, confirming data observed by Daudt et al. (2000) and Viana et al. (2005). Differently from IMI and BUP, after a three days treatment, HCP decrease cortical but not serum corticosterone levels. It should be remarked that, under acute treatment with HCP, there was no significant difference in the corticosterone levels between mice submitted or not to a FST. However, this does not represent a true protection against stress since animals treated with HCP already presented hormone basal levels somewhat higher than animals receiving other treatments. Our results are in agreement with those reported by Franklin et al. (2000) and Webber et al. (2006) for H. perforatum extracts, an effective antidepressant (Linde et al., 1996; Butterweck et al., 1997; Müller et al., 1998; Brenner et al., 2000; Schrader, 2000; Müller, 2003). They showed that acute (Franklin et al. 2000; Webber et al., 2006) or two weeks (Webber et al., 2006) treatment with H. perforatum extracts causes an elevation of plasma and brain corticosterone. Furthermore, several studies report elevation in plasmatic HPA axis hormones in men and animals after antidepressant treatment with citalopram, fluoxetine, reboxetine, mirtazepine, H. perforatum extract WS 5570 and LI 160 (Seifritz et al., 1996; Duncan et al., 1998; Schule et al., 2004a, b; Webber et al., 2006). Some authors support that monoamines elevation in the synaptic clef activate of post-synaptic receptors in the hypothalamus and stimulates the secretion of several hormones, postulating that antidepressant’s effect on monoaminergic systems may lead to the normalization of HPA axis through limbic system connections (Raap and van de Kar, 1999; Tafet and Bernardini, 2003; Schule et al., 2004b).

Franklin et al. (2004) showed that reduced brain levels of corticosterone and cortisol were verified in rats fed during two weeks with pellets containing H. perforatum extracts. The authors suggest that H. perforatum extracts reduce intracerebral glucocorticoid concentration possibly by its action to induce the expression of the multiple drug resistance protein P- glycoprotein. Recent works suggest a possible role of membrane steroid transporters, like Multiple Drug Resistance protein P-glycoprotein (MDR-Pgp), in the regulation of glucocorticoids receptors function during antidepressant treatment (Juruena et al., 2004; Pariante et al., 2004). The hypothesis that antidepressants could exert their clinical effects

122 Confidencial 23/3/2007 through a direct modulation of the glucocorticoids receptor was one of the most innovative for the mechanism of action of this class of drugs (Holsboer, 2000; de Kloet et al., 2005). The MDR-Pgp inhibition operated by antidepressants would inhibit glucocorticoids extrusion from the cell, and thus increase the glucocorticoid-mediated negative feedback on the HPA axis (Pariante et al., 2004). We can assume that the results found in this work with tested drugs, i.e. the lack of acute effect of IMI and BUP on serum corticosterone and their reduction in serum and cortical corticosterone after repeated treatments is due to negative feedback enhancement of the HPA axis.

Contrary to antidepressants, H. perforatum extracts and isolated substances, hypericin, hyperforin and flavonoids, are inducers of this glycoprotein at gastro-intestinal levels (Durr et al., 2000; Perloff et al., 2001; Hesseney et al., 2002; Webber et al., 2006). Even, if Webber et al. (2004) demonstrated that H. perforatum extract, as well as hypericin or hyperforin inhibit MDR-Pgp in a human lymphocytic leukaemia cell line and in porcine brain capillary cells (which model blood brain barrier), in their most recent paper (Webber et al., 2006), a two weeks treatment with H. perforatum extract (LI 160) significantly increased the expression of Pgp in mice brain homogenate. In addition, they observed a linear relation between plasma and brain concentrations of corticosterone in mice treated acute and chronically with different antidepressants, indicating passive corticosterone diffusion into the brain and minor influence of Pgp. The authors support that antidepressant effect on HPA axis should be via monoaminergic transmission. The HPA axis receives and integrates many inputs indicative of stress from different brain areas that converge in the paraventricular nucleus of hypothalamus (Chrousos and Gold, 1998; Reul and Holsboer, 2002). The cooperative interplay between HPA axis and central nervous system provides the means through which thoughts and emotions may regulate hormone secretion (Tafet and Bernadini, 2003).

At present, the mechanism by which HCP reduces brain corticosterone levels remains to be elucidated. Although the possibility that HCP operates via MDR Pgp inhibition cannot be ruled out, the effect of H. caprifoliatum on this protein merits to be performed. It is plausible that ability of HCP to inhibit monoamines uptake could lead to modifications that reduce stress-induced corticosterone rise. In any case, the mechanism by which HCP reduces corticosterone levels seems to be different from the one of antidepressants, since the

123 Confidencial 23/3/2007 repeated treatment with HCP has no effect in non stressed animals and its protection against FST induced corticosterone raise is observed only in the cortex. In order to elucidate the mechanisms involved in HCP effect on corticosterone level, further investigations should be performed.

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CHAPTER 3: Study of antidepressant and antinociceptive activities of Hypericum polyanthemum Klotzsch ex Reichardt

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3.1. INTRODUCTION

In this chapter, we present the study of Hypericum polyanthemum antidepressant profile. As H. caprifoliatum, H. polyanthemum is native to South Brazil and presents high concentration of phloroglucinol derivatives (Nör et al. 2004). Furthermore, some extracts and pure compounds obtained from this species demonstrated monoamine oxidase inhibitory and antinociceptive activities (Gnerre et al, 2001; Viana et al., 2003).

The aim of this chapter was to investigate the potential antidepressant and analgesic effect of H. polyanthemum cyclohexane extract in animal models and search its mode of action by studying the extract effect on the monoaminergic and opioid systems, and its influence on the hypothalamic-pituitary-adrenal (HPA) axis.

3.2. BACKGROUND

Guttiferae is a large family with more than 1,000 species. According to some authors the family is divided into six subfamilies, the Hypericoideae - including the genera Hypericum (tribe Hypericeae), Cratoxylum (tribe Cratoxyleae), Harungana, Psorospermum and Vismia (tribe Vismieae) (Cronquist 1981, Bennet and Lee 1989) - frequently being considered as an independent family by some authors (Robson, 1990).

The genus Hypericum has more than 400 species accommodated in 30 sections (Robson, 1990). The species growing in Rio Grande do Sul, Southern Brazil, belong to the sections Brathys and Trigynobrathys (Robson 1990), the latter with a greater number of native representatives.

The section Brathys includes 88 species of shrubs (rarely small trees), subshrubs, shrublets, and wiry annuals (rarely perennials) distributed into four subsections. This section is predominantly encountered in two centers, Belize and Cuba, and the Venezuela-Colombia

133 Confidencial 23/3/2007 border. From the latter area, the mainly shrubby part has radiated eastward to Roraima, westward to Costa Rica, and southwestward along the Andes and Bolivia, with a disjunct species (H. piriai Arechav.) in southeastern Brazil and Uruguay. Besides H. piriai, the other species from this section found in southern Brazil is H. gentianoides (L.) Britton et al. (Robson,1990).

The section Trigynobrathys comprises 52 species of shrubs, subshrubs, shrublets, as well as perennial and annual herbs. The primary area of speciation has been southeastern Brazil, with a primary radiation (to the south of Amazon) northward to eastern Brazil (Bahia), and to southeastern USA, southward to Uruguay and northern Argentina, westward to Bolivia, Peru, and northern Chile and then north along the Andes to southern Colombia and the Galapagos Islands, and west to New Zealand, Australia, New Caledonia, and New Guinea and to scattered occurrences in south-east Asia. The species of this section are distributed over two subsections: Connatum and Knifa. The species growing in southern Brazil belong to the former (Robson, 1990).

Since 1998, our group studies the chemical and pharmacological features of South Brazilian Hypericum species (Dall’agnol et al 2005, Ferraz et al, 2001; Schmitt, 2001; Daudt et al, 2000; Viana et al, 2003, 2005, 2006). After phytochemical investigation of eight species of Hypericum native to southern Brazil, Ferraz et al. (2002a) verified the absence of hypericin and pseudohypericin in all species. As there is a good correlation between dark glands and hypericin production (Robson, 1990), the absence of hypericins in the Brazilian native species is consistent with the absence of these glands in species from sections Brathys and Trigynobrathys (Robson, 1990, Ferraz et al, 2002a).

South Brazilian Hypericum species have a strong tendency to accumulate phenolic compounds with benzopyran nucleus. Normally, free benzopyrans or chromenes are rare in Hypericum species; these compounds were isolated from few species such as H. revolutum Vahl. (Décosterd et al., 1986). From the chloroform extract of H. polyanthemum aerial parts, three new benzopyrans, 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1); 7- hydroxy-6-isobutyryl-5-methoxy- 2,2-dimethyl-benzopyran (HP2) and 5-hydroxy-6-isobutyryl- 7-methoxy-2,2-dimethyl-benzopyran (HP3) were isolated (Ferraz et al., 2001) (Figure 3.1). A

134 Confidencial 23/3/2007 protocol for in vitro propagation of H. polyanthemum was established by Bernardi e coll (2005). In vitro regenerated plantlets were successfully acclimatized and displayed the same benzopyrans, quantified by HPLC, were the same found in the field-grown plants.

At the same time, the benzopyran nucleus is a common constituent of other phenolic compounds present in Hypericum species, such as benzophenones, xanthones, phloroglucinol derivatives, due to the cyclization of a prenyl radical with a vicinal aromatic hydroxyl group, both frequently found in these substances. Differently from the well-known hyperforin and adhyperforin isolated from H. perforatum, that are polyisoprenilated phloroglucinols, Brazilian species are a source of dimeric structures, consisting of filicinic acid and a phloroglucinol moiety (von Poser et al., 2006). Uliginosin B was isolated from H. myrianthum (Ferraz et al., 2002b), H. carinatum and H. polyanthemum (Nör et al., 2004); hyperbrasilol B from H. caprifoliatum and H. connatum (Nör et al., 2004). A phloroglucinol named HC1 was isolated from H. caprifoliatum but its chemical structure remains not completely elucidated (Viana et al., 2005) (Figure 3.1). From these results we can point out that Southern Brazilian Hypericum species differ chemically from H. perforatum.

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Benzopy rans

O OR1 HP1 R1=Me R2=Me

HP2 R1=Me R2=H

R2O O HP3 R =H R =Me 1 2

Phloroglucinols

O

Uliginosin B (HP4) O OH HO OH

O

OH O

O

O OH HO OH Hyperbrasilol B

O

OH O

O

Putative HC1 HO OH HO OH structure

O

OH O

Figure 3.1.: Molecules isolated from the lipophilic extracts of Southern Brazil Hypericum species.

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With regard to pharmacological activity, especially at the central nervous system (CNS), H. piriai petroleum ether extract, H. caprifoliatum and H. polyanthemum chloroform extract and the benzopyrans (Gnerre et al. 2001) showed in vitro MAOI (monoamino oxidase inhibitory) activity (Gnerre et al. 2001). However, this activity does not appear to be relevant for antidepressant-like effect, since the extracts which displayed this activity were not active in the forced swimming test (FST) (Gnerre et al. 2001). After screening eight Hypericum species using the FST, we had promising results with H. caprifoliatum lipophilic extracts (Daudt et al., 2000; Viana, 2002). H. polyanthemum crude methanolic extract showed slight but not significant reduction in forced swimming test immobility time (Gnerre et al, 2001). On the other hand, H. polyanthemum and H. caprifoliatum cyclohexane extract demonstrated antinociceptive effect in the hot plate and writhing tests (Chapter 1: Viana et al, 2003).

3.3. MATERIALS AND METHODS

3.3.1. Plant material

The aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt were collected in Caçapava do Sul, in the state of Rio Grande do Sul - Brazil (August/2003). The voucher specimens were deposited in the herbarium of the Federal University of Rio Grande do Sul (ICN) (Bordignon1 429).

3.3.2. Preparation of extracts and compounds isolation

The dried and powdered plant material (120 g of aerial parts) was extracted with cyclohexane (plant/solvent ratio 1:10 w/v) by maceration (3x24h), followed by evaporation to dryness under reduced pressure at 45oC yielding an extract termed POL (c.a. 5.0 g). The benzopyrans (HP1, HP2, HP3) and the phloroglucinol derivative, uliginosin B (HP4), were isolated by means of thin layer and column chromatography as described elsewhere (Ferraz et al. 2001; Nör et al., 2004).

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3.3.3. Animals

Adult male Wistar or Sprague Dawley rats (weight 200-300 g) and male CF1 or CD1 Swiss mice (25–30 g) purchased from Fundação Estadual de Produção e Pesquisa em Saúde – RS (Brazil) or IFFA-CREDO/Charles River Laboratories (Domaine des Oncins, Saint-Germain sur L’Arbresle, France) colony were used. The animals were housed five rats or twenty mice in plastic cages (L: 42 cm, W: 27.5 cm, H: 16 cm). All animals were kept under a 12 hours light/dark cycle (lights on at 7:00 a.m.) at constant temperature of 23º ± 1ºC with free access to standard certified rodent diet and tap water.

All behavioural experiments were approved by CONEP - Brazil (National Commission of Research Ethics (Nº project approval number 01-188 at 07/26/2001) and performed according to guidelines of The National Research Ethical Committee (published by National Heath Council – MS, 1998), which are in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

3.3.4. Drugs and Reagents

Desipramine hydrochloride, fluoxetine hydrochloride, imipramine, naloxone, hydrochloride, corticosterone, rabbit antibodies against corticosterone and SCH 23390 (Sigma Aldrich, Saint Quentin Fallavier, France). Cocaine hydrochloride was obtained from la Coopérative Pharmaceutique Française (Melun, France). Cyclohexane, polysorbate 80 (Merck, Darmstadt/ DE), sulpiride (DEG, São Paulo/ BR).

[3H]-dopamine ([3H]-DA, 48 Ci/mmol), [3H]-naloxone (56 Ci/mmol), [3H]-nisoxetine (86 Ci/mmol) and [1,2,6,7(N)]3H-corticosterone (81 Ci/mmol) were purchased from Amersham (Orsay, France). [3H]-noradrenaline ([3H]-NA, 12.5 Ci/mmol), [3H]-serotonin ([3H]-5HT, 25.5 Ci/mmol), [3H]-citalopram (84.2 Ci/mmol) and [3H]-mazindol (24.5 Ci/mmol) were purchased from Perkin-Elmer-NEN Life Science Products (Paris, France). Subsequent dilutions and solutions of these agents were performed in the incubation medium.

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3.3.5. Forced Swimming Test (FST)

In rats: The Porsolt’s procedure (Porsolt et al., 1978) was used with minor modifications. In this test an acrylic box with four sections of 30 x 30 x 40 cm was used. The external walls and the cover were transparent, but the inside sections were dark allowing the isolation of each one of the four quadrants. The rats were submitted to swimming for 15 minutes in water with temperature between 23 ± 2ºC and height of 30 cm (Porsolt et al., employed 15 cm). The ambient temperature was approximately 24ºC. At the end of the swimming exposition, the animals were removed from the water and gently dried. The treatment was administered 5 minutes, 19 and 23 hours after the first swimming exposition. One hour after the last injection (24 hours after the first swimming session), the animals were submitted to a second swimming exposure (5 minutes), and their immobility time was measured.

Rats were treated with POL 270 mg/kg/day, p.o. (3 administrations of 90 mg/kg). This dose was chosen based on results from H. caprifoliatum (Viana et al., 2005). POL was diluted to a concentration of 90 mg/ml in water with 2% of polysorbate 80. Imipramine hydrochloride 20 mg/kg (60 mg/kg/day, p.o.) was used as the antidepressant reference drug and 2% of polysorbate 80 in water was used as the negative control. All treatments were administered at 1 ml/kg body weight.

In mice: The apparatus consisted of a glass cylinder (10 cm internal diameter, 25 cm height) filled with water (19 cm height) at 23 ± 2ºC. Different groups of mice were acutely treated, per os (10 ml/1kg), with POL 180, 270 or 360 mg/kg, uliginosin B 90 mg/kg, imipramine 20 mg/kg, or 2% polysorbate 80 solution in water.

For evaluating the dopaminergic action, different groups were pre-treated with sulpiride (50 mg/kg, i.p.) or SCH 23390 (15 µg/kg, s.c.) 30 minutes before the administration of POL 270 mg/kg p.o. Sixty minutes later, the animals were placed into the cylinder and the total duration of immobility, during a 6 minutes test period, was measured.

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3.3.6. Hot-plate test

The test was performed as described by Viana et al (2003) (Chapter 1). Shortly, the animals were placed on the hot-plate (54 ± 2 ºC) to measure baseline responsiveness, and immediately treated with HP1, HP2, HP3 or HP4 (90 mg/kg, i.p.), the positive control was morphine (6 mg/kg, s.c.) and the negative control was 2 % polysorbate 80 in water (SAL). Treatment-induced changes in responsiveness to heat were observed 30 min later. To evaluate the possible involvement of opioid-mediated mechanisms, groups showing antinociceptive effect were pretreated with naloxone (2.5 mg/kg, s.c.), a nonspecific opioid receptor antagonist, immediately after evaluating baseline responsiveness and 10 min before extract administration.

3.3.7. Locomotor activity

Locomotor activity was accessed automatically in a digiscan photocell activity meter box (45 x 30 x 30 cm) (Omnitech Eletronics Inc, Columbus, OH). The response to POL 270 mg/kg, administered by gavage (10 ml/kg body weight) immediately before the test, was expressed as the number of beams crossed between the 5th and 45th minutes after treatments.

3.3.8. Synaptosomal uptake of [3H]-DA, [3H]-5HT and [3H]-NA

The procedures were identical from Viana et al (2005). Shortly, rats striata (for [3H]-DA uptake), frontal cortex (for [3H]-5HT uptake) and hypothalamus (for [3H]-NA uptake) were dissected out and homogenized in a teflon-glass homogenizer (800 r.p.m.) in 10 volumes (w/v) of ice-cold 0.32 M sucrose solution containing 0.1 mM pargyline. The nuclear material was removed by centrifugation at 1,000 g for 10 minutes, and the supernatant (crude synaptosomal fraction) was used in uptake experiments.

Aliquots (100 µl) of the crude synaptosomal fraction were preincubated for 5 minutes at

37°C in a Krebs-Ringer medium, containing (mM): NaCl 109, KH2PO4 1, CaCl2 1, NaHCO3 27, glucose 5.4, pH 7.4 ± 0.1, in presence or absence of HP4. The incubation was continued

140 Confidencial 23/3/2007 for 5 minutes in the same medium, in the presence of [3H]-DA, [3H]-5HT or [3H]-NA (10 nM; 1 ml final volume). The reaction was stopped by adding 3 ml of ice-cold incubation medium and immediate centrifugation (7,000 g, 10 minutes, 4°C). The pellet was washed with 1 ml of the latter medium and centrifuged in the same condition. The final pellet was sonicated in 250 µl distilled water and aliquots of the homogenate were used for the determination of radioactivity and protein concentrations. The radioactivity was determined by liquid scintillation spectrometry (Tri-carb 2100TR, Packard BioScience Co.) in 4 ml of Ultima Gold (Perkin Elmer). Protein concentrations were determined according to the method of Lowry et al. (1951), using bovine serum albumin as standard.

The specific uptake of DA, 5HT or NA was defined as the difference between the total uptake at 37°C and the non-specific accumulation observed at 0°C, respectively in the presence of 100 µM cocaine for [3H]-DA, 1 µM fluoxetine for [3H]-5HT or 1 µM desipramine for [3H]-NA.

The monoamine synaptosomal uptake was analyzed using the equation of the sigmoidal dose-response curve (variable slope). IC50 and n Hill values were calculated using the equation of the curve-fitting programs Microcal Origin (Microcal Software).

3.3.9. Binding assays to monoamine transporters and opioid receptors

This assay also followed the binding protocol cited by Viana et al (2005; Chapter 1). Dopamine (DAT), noradrenaline (NAT), serotonin (SERT) transporters and opiod receptors binding was assessed, respectively, on striatum, cortex, hypothalamus and forebrain membranes of rats. The crude synaptosomal suspensions, prepared as described in 2.7., were centrifuged at 17,000 g for 30 minutes at 4 °C. The pellet was resuspended by + sonication in: 10 mM Na medium (0.30 mM NaH2PO4, 9.70 mM NaHCO3, pH 7.5 ± 0.1) for the binding to DAT, 50 mM Tris HCl (containing 120 mM NaCl and 5 mM KCl, pH 7.4 ± 0.1) for NAT and SERT or 50 mM Tris HCl pH 7.4 ± 0.1 for opioid receptors; and centrifuged (50,000 g, 10 minutes, 4 °C). The final pellet was resuspended by sonication in the same respective medium for the binding to DAT and SERT, for NAT it was resuspended in 50 mM Tris HCl (containing 300 mM NaCl and 5 mM KCl, pH 7.4 ± 0.1) and for opioid receptors in

50 mM Tris HCl (containing MgCl2 20 mM, pH 7.4 ± 0.1) .

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[3H]-Mazindol (2 nM final concentration) and membranes (100 µg protein) were incubated at 0°C, for 2 h, to evaluate the binding to DAT. For the binding to NAT, 300 µg of protein were incubated with [3H]-nisoxetine (1nM) at 25°C during 2 h; for SERT, 200 µg of protein were incubated with [3H]-citalopram (1 nM) during 1 h at 25°C and for opioid receptors, 400 µg of protein were incubated with [3H]-naloxone (1nM) at 25°C during 2 h. All incubations were carried out in the presence of different HP4 concentrations (3 x 10– 7 – 3 x10– 11 g/ml), in a final volume of 500 µl for monoamine transporters or 1 ml for opioid receptors. Incubations were stopped by dilution with 3 ml of ice-cold incubation medium and immediate filtration through GF/B filters, previously soaked, for at least 1 h, in 0.5% polyethyleneimine. Each tube and filter were rinsed once or twice with 3 ml of ice-cold incubation medium. Filters were counted for radioactivity by liquid scintillation spectrometry (Tri-carb 2100TR, Packard BioScience Co.) in 4 ml of Ultima Gold (Perkin Elmer). The non- specific binding was determined by incubation with cocaine (100 µM, for DAT), desipramine (10 µM, for NAT), fluoxetine (10 µM, for SERT) or naloxone (10 µM, for opioid receptors). Protein concentrations were determined according to the method of Lowry et al. (1951), using bovine serum albumin as standard.

3.3.10. Corticosterone assay

3.3.10.1 Treatments and procedures

Acute treatment: groups of 30 animals were treated by gavage with POL (360 mg/kg), imipramine (IMI 20 mg/kg) or saline (with 10% polysorbate 80). Repeated treatment: groups of 30 animals were treated once daily with the same doses as in the acute treatment, during 3 days.

In each of preceding groups half of the animals (n = 15) were submitted to the forced swimming test (FST), 1h after the single or the last of the three treatments. Mice immobility time was counted in both treatment regimens. All procedures (mice treatment, forced swimming session and decapitation) were performed in separated rooms.

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3.3.10.2. Radioimmunoassay (RIA) Cortical and serum corticosterone levels were determined by radioimmunoassay (RIA). Mice submitted to FST were sacrificed 30 min after the swimming session. Mice not submitted to FST were sacrificed 1h after the last treatment. Trunk blood was collected and was centrifuged twice (13,000 g; 4°C; 15 min). For cortical corticosterone measurements, the frontal cortex of 3 mice was dissected, according to Glowinski and Iversen (1966), weighed (approximately 125 mg) and homogenized in 1 ml phosphate buffer (pH 7.4) containing 1% bovine serum albumin (BSA). All samples were stored at – 40°C until RIA.

Cortex homogenates (1 ml) and serum aliquots (11 µl) were extracted by mixing with 2 ml of ethyl acetate, followed by centrifugation (3,000 g; 4°C; 5 min) to speed up phase separation. Aliquots of 1.5 ml of the ethyl acetate phase were evaporated to dryness. Samples and standards (39 - 10,000 pg/tube) were incubated at 4°C overnight in a final volume of 1 ml with rabbit antibodies against corticosterone and tritiated corticosterone. The separation of free and bound corticosterone was performed, at 4°C, by adding 500 µl of dextran-coated charcoal suspension. Ten minutes later, samples were centrifuged (3,300 g; 4°C; 15 min) and radioactivity contained in the supernatant was measured by liquid scintillation (Tri-carb 2100TR, Packard Bioscience Co).

3.3.11. Statistical analysis The data were evaluated using one or two-way analysis of variance (ANOVA) followed by Student-Newman Keulls test or Student’s t-test depending on the experimental design. P- values less than 0.05 were considered as statistically significant.

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3.4. RESULTS

3.4.1. Forced Swimming Test The cyclohexane extract (POL) reduced the immobility time in both rats and mice tests. POL 270 mg/kg significantly reduced rats immobility time (ANOVA F2,30 = 4.55; p < 0.05) (Figure 3.2).

200

s) 150

( * *

bility 100 o

Imm 50

0 SAL IMI POL

Figure 3.2. Anti-immobility effect of imipramine 60 mg/kg/day, p.o. (IMI) and H. polyanthemum (270 mg/kg/day, p.o.) in rat forced swimming test. The results are presented in mean ± SEM (n = 12-14 rats/group). ANOVA followed by Student Newman-Keuls post-hoc comparisons: *p < 0.05 significant difference in relation to saline (SAL).

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In mice, all tested doses reduced significantly the immobility time (ANOVA, F4,52= 12.13; p < 0.001) (Figure 3.3). The phloroglucinol derivative, uliginosin B (HP4 90 mg/kg), significantly reduced the immobility time when administered in half of POL lowest dose

(180 mg/kg) (Figure 3.4; ANOVA, F 2,29= 15.5; p < 0,001).

300

250 (s) 200 ***

bility 150 o *** *** 100 *** Imm 50

- SAL IMI POL180 POL270 POL360

Figure 3.3. Anti-immobility effect of imipramine 20 mg/kg, p.o. (IMI), and H. polyanthemum cyclohexane extract (POL 180, 270 and 360 mg/kg, p.o.) in mice forced swimming test. The results are presented in mean ± SEM (n = 8–12 mice/group). ANOVA followed by Student Newman-Keuls post-hoc comparisons: ***p < 0.001 compared to saline (SAL).

300 250

(s) 200

bility

o 150 *** ***

Imm 100

50

0 SAL IMI HP4

Figure 3.4. Anti-immobility effect of imipramine 20 mg/kg, p.o. (IMI), and uliginosin B (HP4 90 mg/kg, p.o.) in mice forced swimming test. The results are presented in mean ± SEM (n = 8–12 mice/group). ANOVA followed by Student Newman-Keuls post-hoc comparisons: ***p < 0.001 compared to saline (SAL).

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Three days treatment with POL were effective in reducing the immobility time in FST

(Figure 3.5, F2,44= 20.9; p < 0.001).

250

200

150 *** *** 100

50

0 SAL IMI POL

Figure 3.5. Anti-immobility effect of three days treatment with imipramine 20 mg/kg, p.o. (IMI ) or H. polyanthemum cyclohexane extract (POL, 360 mg/kg, p.o.) in mice forced swimming test. The results are presented in mean ± SEM (n = 15 mice/group). ANOVA followed by Student Newman-Keuls post-hoc comparisons: ***p < 0.001 compared to the respective saline (SAL) group.

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Even though there were no statistically significant differences among POL doses, at 270 mg/kg the anti-immobility effect was more pronounced. Thus this dose was chosen for the pharmacological antagonism test. POL anti-immobility effect in mice was prevented by sulpiride (50 mg/kg) and by SCH 23390 (15 µg/kg) (two away ANOVA, pre- treatment x treatment factor, F2,48 = 7,58; p < 0.01) (Figure 3.6).

300

250

s) ## ( 200 ##

obility 150

Imm ***

100

50

- SAL POL SCH SUL POL + SCH POL + SUL

Figure 3.6. Effect of SCH 23390 (SCH 15 µg/kg, s.c.) or sulpiride (SUL 50 mg/kg, i.p.) administration on the anti-immobility action of POL (H. polyanthemum cyclohexane extract, 270 mg/kg, p.o.) in mice forced swimming test. The drugs were injected 30 minutes before the extract treatment. The results are presented in mean ± SEM (n = 8-12 mice/group). Two way ANOVA followed by Student Newman-Keuls post-hoc comparisons. ***p < 0.001 compared to saline (SAL). ##p < 0.01 compared to POL.

3.4.2 Locomotor activity The treatment with POL 270 mg/kg, p.o., did not significantly modify the locomotor activity of mice, even if we can observe a reduction on mice locomotion. The mean number of beams crossed by control animals was 814 ± 87 while it was 653 ± 94 for POL treated animals.

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3.4.3. Effect of HP4 on the uptake of biogenic amines HP4 inhibited [3H]-DA, [3H]-5HT and [3H]-NA uptake in crude synaptosomal suspensions in a concentration-dependent and monophasic manner (Figure 3.7). It should be 3 pointed out that [ H]-DA uptake (IC50 = 90 ± 38 nM) was more potently inhibited by HP4 than 3 3 [ H]-5HT and [ H]-NA uptake (IC50 = 252 ± 13 nM and 280 ± 48 nM, respectively).

[3H] DA [3H] NA [3H] 5-HT

100

) 80

% ( n

o 60 ti ni

hi n

i 40

take

p 20

U 0

-20 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

HP4 concentration (nM)

Figure 3.7. Effect of HP4 on [3H]-dopamine (•), [3H]-noradrenalin („) and [3H]-serotonin (◊) synaptosomal uptake. The data are presented as percentage of uptake inhibition over basal (mean ± SEM from 4 separated experiments performed in duplicate).

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3.4.4. Effects of HP4 on the binding of uptake inhibitors to monoamine transporters The different tested concentrations of HP4 did not inhibit, in a statistically significant manner, the binding of [3H]-mazindol, [3H]-nisoxetine, [3H]-citalopram to their respective monoamine transporters (Figure 3.8).

3 [ H]-mazindol [3H]-nisoxetina [3H]-citalopram 140

120

g n ) i l 100 o r nd t n

o 80 c f ant bi g i o l

- 60 ] (% H

3 [ 40

20

0 -10 -9 -8 -7 -6 10 10 10 10 10 HP4 concentration (M)

Figure 3.8. Effect of HP4 on [3H]-mazindol, [3H]-nisoxetine, [3H]-citalopram binding to dopamine (DAT), noradrenalin (NAT) or serotonin (SERT) transporters. The data are presented as percentage of uptake inhibition over basal (mean ± SEM from 4 separated experiments performed in duplicate).

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3.4.5 Corticosterone assay

The forced swimming test significantly increased both serum (Fig. 3.9A, Fig. 3.10A) and cortical (Fig. 3.9B, Fig. 3.10B) corticosterone levels measured 30 min later. This corticosterone raise was not prevented neither by acute treatment with imipramine or POL (Figure 3.9). As expected, like HCP (Chapter 2), repeated treatment with IMI or POL significantly reduced the hormone raise induced by FST (Figure 3.10). When compared to CONT or SAL groups imipramine significantly reduced corticosterone levels both in serum and cortex while POL was effective only in reducing cortex levels. The imipramine had no effect in corticosterone levels of non-stressed mice in either treatment regimens whereas, acutely, POL induces an increase in basal levels of serum corticosterone (Figure 3.9A)

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(A) 5050 basal

40 after FST e 40 n ) *** o r m

e ***

u t

s 3030 *** o ser

*** c # rti o 2020 c 100 ml / m ru (µg e 1010 S

00 CONT SAL IMI POL CONT SAL IMI POL

(B) 160 *** ***

e 140

n ***

o *** r 120 e e) t s o

ssu 100

c i ti rt

o 80 c

mg / l g a 60 c i (p rt

o 40

C 20

0 CONT SAL IMI POL

Figure 3.9. Effect of acute per os treatment with imipramine (IMI 20 mg/kg), H. polyanthemum cyclohexane extract (POL 360 mg/kg) or saline (SAL) on serum (A) and cortical (B) corticosterone levels, in mice submitted or not to forced swim test. Data are presented in mean ± SEM (n = 15 mice/group). Two way ANOVA followed by Student- Newman-Keuls post hoc comparisons: ***p < 0.001 compared to the respective non stressed group; # p < 0.05 compared to the respective control groups.

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(A) basal

50 after FST

e *** *** n 40 ** o r m) u r ste e s

co 30 ti r 0 ml o

c ** 10

/ 20 m ## u

r (µg e S 10

0 CONT SAL IMI POL

(B) 160 *** 140 *** e

ron 120 ) e t ue s

s 100 o

s c i i **

rt 80 ## g t o ## c m / l 60 g a c i (p

rt 40 o

C 20 0 CONT SAL IMI POL

10

Figure 3.10. Effect of repeated treatment (3 days) with imipramine (IMI 20 mg/kg, p.o.), H. polyanthemum cyclohexane extract (POL 360 mg/kg, p.o.) or saline (SAL) on serum (A) and cortical (B) corticosterone levels in mice submitted or not to forced swim test. Data are presented in mean ± SEM (n = 15 mice/group). Two way ANOVA followed by Student- Newman-Keuls post hoc comparisons: **p < 0.01, ***p < 0.001 compared to respective non stressed group; #p < 0.01, compared to the respective saline (SAL) group.

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3.4.6. Antinociceptive effect

In order to continue the study of H. polyanthemum antinociceptive effect reported in the Chapter 1 (Viana et al., 2003) we tested the main substances isolated from the cyclohexane extract, benzopyrans (HP1, HP2, and HP3) and uliginosin B (HP4). The antinociceptive effect was observable only in mice treated with HP4 (90 mg/kg, i.p.) (Figure 3.11. One away

ANOVA, F5,66 = 10.44; p < 0.001).

35 *** 30

) s

( 25

y * 20

Latenc 15

10

5

0 SAL MOR HP1 HP2 HP3 HP4

Figura 3.11: Antinociceptive effect of morphine (MOR 10 mg/kg, s.c.), benzopyrans HP1-3 and uliginosin B (90 mg/kg, i.p.) in the hot plate test. Results are presented in mean ± SEM (n = 8–12 mice/group). One way ANOVA followed by

Student Newman-Keuls post-hoc comparisons.: ***p < 0.001, * p < 0.05 compared to saline (SAL).

Contrary to POL, pretreatment with naloxone did not prevent HP4 antinociceptive effect

(two away ANOVA, pre-treatment x treatment factor, F2,41 = 24.98; p < 0.001) (Figure 3.12). This indicates that, although HP4 may be responsible for POL’s analgesia, it does not interact with opioid receptors. The binding assay also indicates that H. polyanthemum antinociceptive effect is not due to a direct action on opioid receptors, since HP4 did not affected [3H]- naloxona binding in membrane preparations (Figure 3.13).

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35

30 ***

25 ** 20 *

15 ### 10 5

0 SAL MOR N+MOR HP4 N+HP4

Figure 3.12. Effect of naloxone (2.5 mg/kg, s.c.) administration on the antinociceptive effect of HP4 (90 mg/kg,i.p.) and morphine (6 mg/kg, s.c.) in the hot plate test. The opioid antagonist was injected 10 minutes before the other treatments. Results are presented in mean ± SEM (n = 8–12 mice/group). Two way ANOVA followed by Student Newman-Keuls post-hoc comparisons.: ***p < 0.001, * p < 0.05 compared to saline (SAL), ###p < 0.01 compared to MOR.

140

120 g

n i d

) n l i 100 b

e

n 80 o x o l a 60 % of contro n ( - ] naloxone H

3 40 HP4 [ 20

0 12-11 34--10 59 6-8 78-7 9- 6 110 210345610 10 10 109 Ligant concentration (M)

Figure 3.13. Effect of naloxone or HP4 on [3H]-naloxone binding to opioid receptors. The data are presented as percentage of uptake inhibition over basal (mean ± SEM from 4 separated experiments performed in duplicate).

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3.5. DISCUSSION

In this work we carried on the pharmacological investigation of Hypericum species, focusing on the potential antidepressant and antinociceptive effect of H. polyanthemum cyclohexane extract (POL) and isolated compounds. The results demonstrated that POL has antidepressant-like effect in the forced swimming test (FST) both in rats and mice, that is sustained after repeated treatment (3 days). The reduction in immobility time is not related to motor stimulating activity, which is important to differentiate antidepressants from stimulants in the FST (Bourin, 1990; Porsolt et al., 1991). Moreover, pre-treatment with dopaminergic receptors antagonists, SCH 23390 and sulpiride prevented POL antiimmobility effect demonstrating an involvement of dopaminergic system in the FST activity. Since uliginosin B (HP4) at 90 mg/kg was as effective in the FST as POL 360 mg/kg and inhibited monoamine uptake, we presume that HP4 can be the molecule responsible for the POL antiimmobility effect. In addition, HP4 demonstrated antinociceptive effect in the hot-plate test, whereas the other major substances (benzopyrans) present in the cyclohexane extract were inactive.

Research groups working with Hypericum species still debate on the substance or substances responsible for antidepressant effect. Some authors support that H. perforatum hypericin and flavonoids are the main antidepressant constituents (Butterweck et al., 1998 and 2004) and others say that active substances are the phloroglucinol derivatives, hyperforin and ad-hyperforin (Chatterjee et al., 1998a; Muller et al., 2003; Mennini and Gobbi, 2004; Wurglics and Schubert-Zsilavecz, 2006). Our results confirm that, as hyperforin, HP4 and H. caprifoliatum phloroglucinol, HC1, exhibit potential antidepressant activity and are very likely to be the substance with antidepressant-like activity. This assumption is reinforced by the fact that cyclohexane extracts are devoid of flavonoids, which remain in the hydrophilic extracts (Dall’agnol et al, 2003) and Southern Brazilian Hypericum species do not have hypericin producing glands (Robson, 1990, Ferraz et al, 2002a).

Besides studying H. polyanthemum antidepressant effect via monoaminergic system, we investigated its influence on serum and cortex corticosterone levels. Disturbances in mood often coincide with abnormal levels of corticosteroids. It has been observed that the

155 Confidencial 23/3/2007 hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis observed in depressed patients was corrected by clinically effective treatments using classical antidepressant drugs (Barden et al., 1995; Gold et al., 1995; Holsboer and Barden, 1996; Pariante et al., 2004). In the Chapter 2, we demonstrated that 3 days treatment with imipramine or bupropion reduce FST-induced corticosterone levels raise in serum and brain. However, H. caprifoliatum cyclohexane extract reduced corticosterone levels increase in brain but not in serum. POL effects were similar to that of H. caprifoliatum, i.e. 3 days treatment with the extract affects only cortical corticosterone levels in mice submitted to FST. In addition, in non stressed mice acute treatment causes corticosterone levels raise in cortex and serum, but only the later is significant. Curiously we can observe a tendency of IMI in reducing serum and cortex corticosterone levels in non-stressed mice. The ability of antidepressants in reducing basal levels of corticosterone has been already reported (Shimoda et al., 1988; Duncan et al., 1998). Taken together these data corroborate with the idea of a diverse mechanism of action for Hypericum extracts.

The possibility of a mechanism of action different from classical antidepressants has already been proposed by us for Brazilian Hypericum species (Chapter 1: Viana et al., 2005; 2006) and by others for H. perforatum extracts (Gobbi et al., 2001; Roz et al., 2002; Yoshitake et al., 2004). Hyperforin as well as HP4 and HC1 inhibit monoamines uptake without binding to their respective transporters (Chatterjee et al., 1998b; Müller et al., 1997, 1998; Jensen et al., 2001), and affect corticosterone levels in a different manner than antidepressants. Franklin et al. (2004) also observed reduction only in cortical levels of corticosterone. In Chapter 1 (Viana et al., 2005) we raised the possibility that the mechanism of action could be related to the inhibition of neuronal monoamine uptake, most potently of dopamine. This effect could be caused by an indirect effect on monoamine transporters, most likely due to the elevated neurotransmitter levels in the synaptic clefts, thus leading to an alteration of monoamine transporter function different from the mechanism of action of reference monoamine uptake inhibitors.

Although the mechanism by which the activation of monoaminergic system occurs is not elucidated yet, this effect seems to be responsible not only for the antidepressant-like

156 Confidencial 23/3/2007 activity but also for the antinociceptive effect. This hypothesis was considered since HP4 activity in the hot-plate test is not prevented by naloxone and both HP4 and HC1 do not inhibit [3H]-naloxone binding to opioid receptors (Chapter 1: Viana et al., 2003; 2006).

Antidepressants, mainly noradrenergic (tricyclics and venlafaxine), are widely prescribed for the treatment of chronic and neuropathic pain (Brandão, 1997; Schereiber et al., 1998; Atkinson et al., 1999; Lynch, 2001). The nature and underlying mechanisms of antidepressant analgesia is not clear, there is evidence showing that antidepressants may induce endogenous opioid peptides, and that this effect is independent of their antidepressant effect (Gray et al., 1998; Atkinson et al., 1999). Besides noradrenergic neurons, studies evidence that large neurons of the dopaminergic network of the PAG (periaquedutal grey) participate in supraspinal nociceptive responses after opiates treatment through the involvement of D1 dopamine receptors (Flores et al., 2004). On the other hand, some authors support that the analgesic effect of antidepressants like nomifensina and dopaminergic agonists (amphetamine, apomorphine, bromocriptine) are related only to the dopaminergic system (Michael-Titus et al., 1990; Morgan and Franklin, 1991; Frussa-Filho et al., 1996; Altier and Stewart, 1999; Gilbert and Franklin, 2001). Several studies suggest that the activation of mesolimbic dopamine (DA) neurons arising from the cell bodies of the ventral tegmental area (VTA) and projecting to the nucleus accumbens plays an important role in mediating the suppression of tonic pain (Altier and Stewart, 1999). Therefore since HP4 and HC1 monoamine uptake inhibition is somewhat more potent to dopamine, it is possible that the dopaminergic system is involved in the antinociceptive effect of HCP and POL. Other experiments will be necessary to confirm this hypothesis.

Finally, this study highlights the importance of the accurate knowledge on plants chemical constitution in previewing pharmacological effects. In a first screening procedure several Hypericum total methanol extracts were tested in the FST and H. polyanthemum have not shown antidepressant like effect (Gnerre et al., 2001). After chemical analysis of Southern Brazilian Hypericum species and identification of analogous phloroglucinols in lipophilic extracts of H.caprifoliatum, that have already demonstrated antidepressant-like activity (Daudt et al., 2000), and H. polyanthemum (Nor et al., 2004), we investigated the potential

157 Confidencial 23/3/2007 antidepressant activity of H. polyanthemum cyclohexane extract. The results obtained in this study are in accordance with our previous reports regarding the effect of lipophilic extracts of H. caprifoliatum (Chapter 1; Chapter 2). Thus we can point out that the phloroglucinol derivatives represent a molecular pattern promising to the development of antidepressants with innovative mechanism of action.

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JENSEN, A. G.; HANSEN, S. H.; NIELSEN, E. O. Adhyperforin as contributor to the effect of Hypericum perforatum L. in biochemical models of antidepressant activity. Life Sciences, v.68, p.1593-1605, 2001. LOWRY, O.H.; ROSENBROUGH, N.J.; FARR, A.L.; RANDALL, R.J. Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry, v.193, p.265-275, 1951. LYNCH, M. E. Antidepressants as analgesics: a review of randomized controlled trials. Journal of Psychiatry & Neuroscience, v. 26, n.1, p.30-36, 2001. MENNINI, T.; GOBBI, M. The antidepressant mechanism of Hypericum perforatum. Life Sciences, v.75, n.9, p.1021-1027, 2004. MICHAEL-TITUS, A.; BOUSSELMAME, R.; COSTENTIN, J. Stimulation of dopamine D2 receptors induces an analgesia involving an opioidergic but non enkephalinergic link. European Journal of Pharmacology, v.187, n.2, p.201-207, 1990. MORGAN M.J., FRANKLIN K.B.J. Dopamine receptor subtypes and.317-322 formalin test analgesia. Pharmacology Biochemistry And Behavior . v.40, n.2, p, 1991. MÜLLER, W.E.; ROLLI, M.; SCHAFER, C.; HAFNER, U. Effects Hypericum extract (LI 160) in biochemical models of antidepressant activity. Pharmacopsychiatry, v. 30, p.102-107, 1997. MÜLLER, W.E.; SINGER, A.; WONEMANN, M.; HAFNER, U.; ROLLI, M.; SCHAFER, C. Hyperforin represents the neurotransmitter reuptake inhibiting constituent of Hypericum extract. Pharmacopsychiatry, v.31, p.16–21, 1998. MÜLLER, W.E. St John's wort research from mode of action to clinical efficacy. Pharmacological Research 47,101-9, 2003. NÖR, C.; ALBRING D.; FERRAZ, A.B.F.; SCHRIPSEMA , J.; PIRES ; V.; SONNET, P.; GUILLAUME, D. ; VON POSER, G.L. Phloroglucinol derivatives from four Hypericum species belonging to the Trigynobrathys section. Biochemical Systematics and Ecology v.32, p.517- 519, 2004. PARIANTE, C. M.; THOMAS, S. A.; LOVESTONE, S.; MAKOFF, A.; KERWIN, R. W. Do antidepressants regulate how cortisol affects the brain? Psychoneuroendocrinology, v.29, p. 423-447, 2004. PORSOLT, R.D.; ANTON, G.; BLAVET, N.; JAFRE, M. Behavioral despair in rats, a new model sensitive to antidepressant treatments. European Journal of Pharmacology, v.47, p.379-391, 1978. PORSOLT, R. D.; LENÈGRE, A.; MCARTHUR. Pharmacological Models of Depression. Animal Models in Psychopharmacology, p. 139, 1991. ROBSON, N.K.B. Studies in the genus Hypericum L. (Guttiferae) 8. Sections 29. Brathys (part 2) and 30. Trigynobrathys. Bulletin of the British Museum of Natural History, v.20, p.1-151, 1990. ROZ, N.; MAZUR, Y.; HIRSHFELD, A.; REHAVI, M. Inhibition of vesicular uptake of monoamines by hyperforin. Life Sciences, v.71, p.2227-2237, 2002. SCHMITT, A.C.; RAVAZZOLO, A.P.; VON POSER, G.L. Investigation of some Hypericum species native to Southern of Brazil for antiviral activity. Journal of Ethnopharmacology, v.77, p.239-245, 2001. SCHREIBER, S.; GETSLEV, V.; WEIZMAN, A.; PICK, C. G. The antinociceptive effect of moclobemide in mice is mediated by noradrenergic pathways. Neuroscience Letters, v. 253, p. 183–186, 1998.

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SHIMODA, K.; YAMADA, N.; OHI, K.; TSUJIMOTO, T.; TAKAHASHI, K.; TAKAHASHI, S. Chronic administration of tricyclic antidepressants suppresses hypothalamo-pituitary- adrenocortical activity in male rats. Psychoneuroendocrinology, v.13, n.5, p.431-440, 1988. VIANA, A. F. Estudo da atividade psicofarmacológica de espécies de Hypericum nativas do Rio Grande do Sul e toxicidade aguda de Hypericum caprifoliatum Cham. & Schledt. Dissertação de Mestrado, UFRGS, 2002. 124p VIANA A.F., HECKLER A.P., FENNER R., RATES S.M., Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae). Brazilian Journal of Medical and Biological Research, v.36, p.631-634, 2003. VIANA, A.; DO REGO, J.C.; VON POSER, G.; FERRAZ, A.; HECKLER, A.P.; COSTENTIN, J.; KUZE-RATES, S. M. The antidepressant-like effect of Hypericum caprifoliatum Cham & Schlecht (Guttiferae) on forced swimming test results from an inhibition of neuronal monoamine uptake. Neuropharmacology, v. 49, p.1042-1052, 2005. VIANA, A.; DO REGO, J.C.; MUNARI, L.; DOURMAP, N.; HECKLER, A. P.; DALLA COSTA, T.; von POSER. G. L.; COSTENTIN, COSTENTIN, J.; RATES, S. M.K. Hypericum caprifoliatum (Guttiferae) Cham. & Schltdl.: a species native to South Brazil with antidepressant-like activity. Fundamental & Clinical Pharmacology, v.20, p.507–514, 2006. VON POSER, G.L.; RECH, S.B.; RATES, S.M.K.. Chemical and Pharmacological Aspects of Southern Brazilian Hypericum Species. Floriculture, Ornamental and Plant Biotechnology Volume IV, p. 509-515, 2006. WURGLICS, M.; SCHUBERT-ZSILAVECZ, M. Hypericum perforatum: a 'modern' herbal antidepressant: pharmacokinetics of active ingredients. Clinical Pharmacokinetics, v.45, n.5, p.449-468, 2006. YOSHITAKE, T.; IIZUKA, R.; YOSHITAKE, S.; WEIKOP, P.; MULLER, W. E.; OGREN, S. O.; KEHR, J. Hypericum perforatum L (St John's wort) preferentially increases extracellular dopamine levels in the rat prefrontal cortex. British Journal of Pharmacology, v.142, n.3, p.414- 418, 2004.

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Casa

CHAPTER 4: Effect of Hypericum caprifoliatum and Hypericum polyanthemum cyclohexane extracts on monoamine and opioid receptor-stimulated [35S]GTPγS binding in rat brain membranes.

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4.1. INTRODUCTION

In previous chapters we demonstrated that Hypericum caprifoliatum (HCP) and H. polyanthemum (POL) cyclohexane extracts have potential antidepressant and antinociceptive effects (Chapter 1 and 3). The extracts and their main phloroglucinol derivatives (HC1 and HP4) act on pre-synapse by inhibiting monoamine uptake but differently from antidepressants, they did not bind to the monoamine transporters. Furthermore, the conventional antidepressant like-effect in the forced swimming test was inhibited by D1 and D2 antagonists while the antinociceptive effect was abolished by naloxone, a nonselective opioid receptor antagonist. These latter observations suggest an action also at post synaptic level.

In this chapter we deal with a functional binding approach for investigating post synaptic effects. We investigated the effect of acute or repeated administration of H. caprifoliatum and H. polyanthemum cyclohexane extracts on immobility time in FST and on monoamine and opioid receptor-stimulated [35S]GTPγS binding in rat brain membranes. We have also investigated the effect of HC1 and HP4 on mice FST and [35S]GTPγS binding assay.

4.2. BACKGROUND

To date, all the accumulated knowledge about mood disorders treatments (including non medication ones) did not provide a unique and definitive explanation for the antidepressant action (Duman et al., 1997; Nestler, 1998; Stahl, 2000) and it also still lacks a clear understanding of the neural substrates that are abnormal in depression and related syndromes (Nestler and Carlezon, 2006). Biochemical research in mood disorders has focused, along the cascade of events involved in signal transduction, from studies at the level of the primary messenger, the monoamine neurotransmitter, to the level of the neurotransmitter receptors, and lately to information transduction mechanisms beyond receptors, involving the coupling of receptors with signal transducers (Schatzberg and Schildkraut, 1995; Duman et al., 1997; Gould and Manji, 2002).

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Targeting multiple neurochemical mechanisms such as serotonin, noradrenaline and dopamine systems in the brain region is hypothesized to be part of the mechanism underlying the efficacy of many conventional antidepressants, including tricyclic antidepressants, monoamine oxidase inhibitors and selective serotonin reuptake inhibitors in the treatment of depression (Millan et al., 2006; Pacher et al., 2001; Chen and Lawrence, 2003). However, it is well known that all antidepressants need a delay of at least two weeks to elicit their therapeutic effect in human, while the raise in monoamines is immediate; therefore, adaptive changes in the monoaminergic system are believed to underlie the therapeutic effectiveness of a variety of antidepressant drugs. It has been suggested that one of such adaptations is the desensitization of monoamine receptors (Blier and de Montigny, 1994; Le Poul et al., 2000; Avissar and Schreiber, 2006). The conflicting observations regarding the regulation of monoamine receptor sensitivity following antidepressant treatment may be due to the effects of these drug treatments on complex neuronal circuits, or differences in the regulation of monoamine receptors function in specific brain regions.

The monoaminergic receptors belong to the superfamily of heptahelical G protein coupled receptors (GPCRs). It means that they have the capacity to activate guanine nucleotide-binding proteins (G proteins), localized in the inner surface of the plasma membrane (Gould and Manji, 2002). G proteins are a ubiquitous family of heterotrimeric proteins, formed by α,β and γ subunits. They have a crucial role as information transducers across the plasma membrane, coupling receptors to various effectors. It is estimated that about 80% of all known hormones, neurotransmitters, and neuromodulators elicit cellular responses through G proteins coupled to a variety of intracellular effectors (Chen et al., 1999; Hermans, 2003).

Growing clinical and pre-clinical evidence suggest that GPCR and its regulation, may be involved in both the pathogenesis and treatment of mood disorders (for review, see Avissar and Schreiber, 2002; Gould and Manji, 2002; Schreiber and Avissar, 2003). The available evidence relies on: (i) effect of lithium on receptor-G protein coupling (Avissar and Schreiber, 1992; Donati et al., 2001; Shen et al., 2002; Pejchal et al., 2002); (ii) differential alterations in the concentration and/or function of G proteins of patients with bipolar disorder, major depression and seasonal affective disorder (for review, see Avissar and Schreiber, 2002;

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Schreiber and Avissar, 2003); (iii) alterations in the levels of G proteins in postmortem tissues of patients with mood disorders (Young et al., 1991, 1993; Friedman and Wang, 1996); (iv) findings concerning G protein encoding genes, p.ex. G 3, as susceptibility locus for major depressive disorder and seasonal affective disorder and as an indicator for antidepressant treatment response (Lee et al., 2004); (v) evidence that activated Gα subunit-adenylyl cyclase coupling plays a major role in mediating the actions of chronic antidepressant treatment, including eletroconvulsive shock (Ozawa and Rasenick, 1989; Gould and Manji, 2002; Donati and Rasenick, 2003, 2005). Antidepressants effects on G-protein functionality are variable depending of the protocol, brain area, antidepressant and duration of treatment. Chronic 35 treatment with fluoxetine and imipramine reduces 5-HT1A receptor-mediated [ S]GTPγS binding, in dorsal raphe (Hensler, 2002; Pejchal et al., 2002; Castro et al., 2003) and lateral septum (Shen et al., 2002) but not in striatum or hippocampus (Pejchal et al., 2002). Others studies have shown an increase in Gαs coupling to the catalytic subunit of adenylate cyclase (Chen and Rasenick, 1995; Chen et al., 1999). Finally, some studies have shown no change in G protein subunit levels following chronic treatment with desipramine or amitriptyline (Emamghoreishi et al., 1996. Dwivedi and Pandey,1997; Hensler, 2002).

A method that allows measuring agonist effect on the GPCR is the [35S]GTPγS binding assay to brain structures. This assay is considered convenient, easy, quick and quite accurate to measure the level of G protein activation following agonist occupation of a GPCR (Lazareno, 1999). Since Gα subunit holds GTPase activity, the endogenous GTP is replaced by its nonhydrolyzable analog [35S]-guanosine-5’-O-(3-thio) triphosphate, that irreversibly binds to Gα (Bymaster et al., 2001; Harrison and Traynor 2003). This assay allows the construction of concentration-effect curves and therefore potency (EC50) and relative efficacy

(Emax) measures, with the advantage that agonist measures are not subjected to amplification or other controls that have to be considered when measuring downstream systems, such as adenylyl cyclase, phospholipase C or gene expression, and so provide the best source of information about ligand-induced events at the receptor. Although this assay determines the degree of binding of [35S]GTPγS it is truly a functional assay because it is the activation of a receptor by an agonist that induces [35S]GTPγS binding to Gα subunit (Harrison and Traynor 2003).

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4.3. MATERIALS AND METHODS

4.3.1. Plant material

The aerial parts of Hypericum caprifoliatum Cham. and Schltdl. were collected in the region of Viamão, while the ones from Hypericum polyanthemum Klotzsch ex Reichardt were collected in the region of Caçapava do Sul, both in the state of Rio Grande do Sul - Brazil. The voucher specimens were deposited in the herbarium of the Federal University of Rio Grande do Sul (ICN) (Bordignon 1496 and 1429, respectively).

4.3.2. Preparation and purification of the extract

H. caprifoliatum (HCP) and H. polyanthemum (POL) extracts were prepared as described by Viana et al. (2003, 2005). Shortly, the dried and powdered plant material was extracted with cyclohexane (Merck, Darmstadt/ DE) followed by evaporation purification (wax elimination). Thin layer and column chromatography were used to isolate the phloroglucinol derivative fraction, HC1 from H. caprifoliatum and HP4 from H. polyanthemum.

4.3.3. Animals

Adult male Sprague Dawley rats, weighing 180-200 g, were purchased from IFFA- CREDO/Charles River Laboratories (Domaine des Oncins, Saint-Germain sur L’Arbresle, France). Male CF1 mice (25–30 g) were purchased from Fundação Estadual de Produção e Pesquisa em Saúde – RS (Brazil). The animals were housed by five rats or ten mice in Makrolon cages (L : 42 cm, W : 27.5 cm, H : 18 cm). All animals were kept under a 12 hours light/dark cycle (lights on between 7:00 hour and 19:00 hour) in a well ventilated room, at constant temperature of 22 ± 1ºC, with free access to standard certified rodent diet and tap water ad libitum.

All the behavioral experiments were performed according to guidelines of The Brazilian Research Ethical Committee (published by National Heath Council – MS, 1998), which are in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

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4.3.4. Treatments

For the GTP assays, groups of rats (n=4) were submitted to 4 different treatment regimens: T1) one treatment by oral gavage 1 h before sacrifice; T2) three treatments during 24h (23, 5 and 1 h before sacrifice); T3) two treatments per day during 5 days; T4) two treatments per day during 5 days followed by a 3 days wash-out before assays. The extracts were dissolved in water with 5% polisorbate 80 to a dose of 90 mg/kg.

4.3.5. Forced Swimming Test (FST)

FST was performed as previously reported (Viana et al., 2005). In brief, before treatment beginning rats were submitted to swimming for 15 minutes in water 30 cm depth with temperature between 23 ± 2ºC. The ambient temperature was approximately 22ºC. At the end of the swimming exposition, the animals were removed from the water and gently dried. One hour after the last treatment (T2, T3) or three days after treatment discontinuation (T4), the animals were submitted to a second swimming exposure (5 minutes), and their immobility time was measured.

In mice, the FST apparatus consisted of a glass cylinder (10 cm internal diameter, 25 cm height) filled with water (19 cm height) at 23 ± 2ºC. The animals were acutely treated with HC1, HP4 (90 mg/kg) or 2% polysorbate 80 solution in water, per os (10 ml/1kg). One hour later (T1), the animals were placed into the cylinder and the total duration of immobility, during a 6 minutes test period, was measured.

4.3.6. [35S]GTPγS binding assay

Crude membrane preparations were isolated according to the modified method described elsewhere (Viana et al., 2005). Rats were sacrificed by decapitation one hour after the treatment for T1 and last treatment T2 and T3, or three days later for T4. Frontal cortex, striatum, hypothalamus and thalamus were removed and homogenized in 20 vol. of 0.32M sucrose. The same brain structures were removed from non-treated rats and used for direct incubation with HC1 or HP4. The homogenates were centrifuged (1,000g for 15 min); the

169 Confidencial 23/3/2007 supernatants from two centrifugations were combined and centrifuged (17,000g for 30 min). The resulting pellet was then suspended in Tris buffer (50 mM Tris/HCl, 100 mM NaCl, 5 mM

MgCl2, and 1 mM EDTA, pH 7.4), sonicated, and centrifuged (50,000g for 10 min). The final protein concentration was determined by the method of Lowry (Lowry et al. 1951).

[35S]GTPγS binding assays were performed as follows. Membranes (15-20µg/100µl) were incubated at 25ºC for 2 h in the assay buffer (Tris buffer added with 1mM dithiothreitol (DTT) and 0,1% metabisulphite) with 10 µM GDP (Sigma–Aldrich Co.), 10-4-10-8 M of dopamine (DA), noradrenalin (NA) and serotonin (5-HT), 10-5-10-9 of DAMGO (Sigma–Aldrich Co.), or 10-6-10-10 M of HC1 or HP4, in the presence of 0.1 nM [35S]GTPγS (1250 Ci/mmol; Perkin-Elmer, Courtaboeuf, France) in a total volume of 1 ml. Basal binding was assessed in the absence of peptide analogue and presence of GDP, and the non-specific binding was assessed in the presence of 10 µM guanosine-5’-O-(3-thio)triphosphate (GTPγS; Sigma– Aldrich Co.). The entire mixture was incubated at 25 ºC for 2 h and filtered through Whatman GF/B glass fiber filters, which had been pre-soaked for 2 h in Tris buffer, and washed three times with 4 ml of ice-cold Tris buffer, using a Millipore Sampling Manifold (Billerica, MA, USA). Bound radioactivity was determined in Tri-Carb 2100TR liquid scintillation counter (Packard) after overnight extraction of the filters in 4 ml of Ultima Gold scintillation fluid (Perkin-Elmer). Four independent experiments for each assay were carried out in duplicate.

4.3.7. Data analysis

Data are expressed as means ± SEM. The percent stimulation of [35S]GTPγS binding was calculated according to the following formula: (S - B)/B x 100%, where S is the stimulated level and B is the basal level of [35S]GTPγS binding in the presence of GDP. Individual dose–response curves were obtained by a non-linear regression analysis.

The data were evaluated using one analysis of variance (ANOVA) followed by Student- Newman Keulls test or Student’s t-test depending on the experimental design. P-values less than 0.05 were considered as statistically significant.

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4.4. RESULTS

4.4.1. Forced Swimming Test

All treatment regimens with HCP significantly reduced immobility time (One way

ANOVA, F5,53 = 17.46). Five days treatment (T3) was not more effective than the classical Porsolt protocol, i.e. 3 treatments within 24h (T2). The antiimobility effect remains even after 3 days wash-out (T4) although less pronounced than those observed in T2 and T3 (Figure 4.1).

Similarly to HCP all treatment regimens with POL were effective (One way ANOVA,

F5,54 = 4.89). Even though there are no statically significant differences between POL and HCP treatments it can be observed that the POL effects are weaker than those of HCP (Figure 4.1).

250 T2 T3 T4

200 * (s) * 150 * * * * * bility * * o * * 100

Imm 50

- SAL HCP POL

Figure 4.1: Effect of T2 (three treatments during 24h), T3 (two treatments per day during 5 days) and T4 (two treatments per day during 5 days followed by a 3 days wash-out) with 90 mg/kg of H. caprifoliatum (HCP), H. polyanthemum (POL) or saline (SAL 1ml/kg) on rat forced swimming test. Data are presented in mean ± SEM (n = 8 rats/group). Significantly different values were detected by one way ANOVA followed by post hoc Student-Newman-Keuls test : *p < 0.05; **p < 0.01; ***p < 0.001 as compared to their respective saline (SAL) group.

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Since the phloroglucinols isolated from H. caprifoliatum (HC1) and from H. polyanthemum (HP4) dose dependently inhibited monoamines uptake (as shown in Chapter 1), here we tested them in the FST to investigate if they have in vivo effect. Both HC1 and

HP4 were effective in reducing mice immobility time (One way ANOVA, F2,27 = 12.36) (Figure 4.2).

250

) 200 s *** 150 ***

bility ( 100 mo

Im 50

0 SAL HC1 HP4

Figure 4.2: Effect of acute treatment with the phloroglucinols HC1 (H. caprifoliatum) and HP4 (H. polyanthemum) 90 mg/kg p.o.; or saline (SAL 100 ml/kg) on mice forced swimming test. Data are presented in mean ± SEM (n = 10 mice/group). Significantly different values were detected by one way ANOVA followed by post hoc Student-Newman-Keuls test : ***p < 0.001 as compared to their respective saline (SAL) group.

4.4.2. [35S]GTPγS binding assay

Ex vivo experiments

The different treatment regimens induced contrasting results. Treatments T1 and T2 35 with HCP and POL increased the maximal effect (Emax) of [ S]GTPγS binding induced by all monoamines (Figures 4.3 and 4.4) and did not affect DAMGO curves features. The 35 monoamine potency (EC50) to induce [ S]GTPγS binding was also increased (Table 4.1).

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Saline HCP POL

100 A 100 C

80 80 n n o o i i

60 t 60 a at ul ul m m i 40 t 40 Sti % S %

20 20

0 0 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 log[DA] (M) log[5-HT] (M) 100 B 100 D

80 80 n n o i o

i

60 at 60 at ul ul m i m i

40 St 40 St % %

20 20

0 0 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 log[NA] (M) log[DAMGO] (M)

Figure 4.3: Effect of T1 (single administration) with HCP, POL (90 mg/kg, p.o.) or saline on agonist-stimulated [35S]GTPγS binding measured ex vivo. Membranes prepared from rat striatum (A), hypothalamus (B), frontal cortex (C) and thalamus (D) were incubated with increasing doses of dopamine (A), noradrenaline (B), serotonin (C) or DAMGO (D) in the presence of 0.1 nM [35S]GTPgS and 10 mM GDP. Data are presented as percentage of binding stimulation over GDP basal stimulation (absence of agonist). Mean ± SEM from 4 separated experiments carried out in duplicate.

.

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Saline HCP POL

120 A 120 C

100 100

n 80

n 80 io io

t t a a l l u u 60 60 im im t t S S

40 % 40 %

20 20

0 0 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 log[DA] (M) log[5-HT] (M) 16 0 B 100 D 140 80 120 n n o i io 100

t 60 at a l ul u 80 m i im t 40 St S 60 % % 40 20 20 0 0 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 log[NA] (M) log[DAMGO] (M)

Figure 4.4: Effect of T2 (three treatments during 24h (23, 5 and 1 h before assay)) with HCP, POL (90 mg/kg, p.o.) or saline on agonist-stimulated [35S]GTPγS binding measured ex vivo. Membranes prepared from rat striatum (A), hypothalamus (B), frontal cortex (C) and thalamus (D) were incubated with increasing doses of dopamine (A), noradrenaline (B), serotonin (C) or DAMGO (D) in the presence of 0.1 nM [35S]GTPgS and 10 mM GDP. Data are presented as percentage of binding stimulation over GDP basal stimulation (absence of agonist). Mean ± SEM from 4 separated experiments carried out in duplicate.

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Repeated treatment (T3 and T4) with HCP or POL induced a reduction in the Emax and 35 potency (EC50) of [ S]GTPγS binding stimulated by of monoamines (Figure 4.5 and 4.6). This reduction was significant for the three monoamines when membranes were prepared one hour after the last treatment (T3) and even following three days wash-out (T4) the it was still observable (Table 4.1). DAMGO-stimulated curves were not affected by the treatments.

Saline HCP POL

100 AC100

80 80 n n o o 60 60 ulati ulati m m i i t t 40

S 40 S % %

20 20

0 0 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 log[DA] (M) log[5-HT] (M)

100 100 B D

80 80

n n o 60 o 60 ati ati l l u u m m 40 40 Sti Sti % % 20 20

0 0 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 log[NA] (M) log[DAMGO] (M)

Figure 4.5: Effect of T3 (two treatments per day during 5 days) with HCP, POL (90 mg/kg, p.o.) or saline on agonist-stimulated [35S]GTPγS binding measured ex vivo. Membranes prepared from rat striatum (A), hypothalamus (B), frontal cortex (C) and thalamus (D) were incubated with increasing doses of dopamine (A), noradrenaline (B), serotonin (C) or DAMGO (D) in the presence of 0.1 nM [35S]GTPgS and 10 mM GDP. Data are presented as percentage of binding stimulation over GDP basal stimulation (absence of agonist). Mean ± SEM from 4 separated experiments carried out in duplicate.

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Saline HCP POL 100 A 100 C

80 80 n n o io

60 t 60 a

l u ulati m im t 40 40 S Sti %

%

20 20

0 0 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 log[DA] (M) log[5-HT] (M) 100 B 100 D

80 80

n n o o i 60 i 60 at at

ul ul m m i i 40 40 St St

% % 20 20

0 0 -8 -7 -6 -5 -4 -3 -10-9-8-7-6-5-4 log[NA] (M) log[DAMGO] (M)

Figure 4.6: Effect of T4 (two treatments per day during 5 days followed by a 3 days wash-out) with HCP, POL (90 mg/kg, p.o.) or saline on agonist-stimulated [35S]GTPγS binding measured ex vivo. Membranes prepared from rat striatum (A), hypothalamus (B), frontal cortex (C) and thalamus (D) were incubated with increasing doses of dopamine (A), noradrenaline (B), serotonin (C) or DAMGO (D) in the presence of 0.1 nM [35S]GTPgS and 10 mM GDP. Data are presented as percentage of binding stimulation over GDP basal stimulation (absence of agonist). Mean ± SEM from 4 separated experiments carried out in duplicate.

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Table 4.1: Effect of HCP and POL different treatment regimens on potency (EC50) and relative efficacy (Emax) of [35S]GTPγS binding.

EC50 (µM) Emax DA NA 5-HT DAMGO DA NA 5-HT DAMGO T1 25 ± 6.2 42.5 ± 3.6 156 ± 25 0.24 ± 0.04 60.6 ± 2.2 48.9 ± 3.9 45.3 ± 3.3 79.7 ± 4.5

T2 18.1 ± 2.4 39.4 ± 6.5 135 ± 26 0.37 ± 0.07 59.3 ± 3.9 47.9 ± 5.9 33.8 ± 2.7 71.6 ± 5.2 Saline T3 5 ± 1 10.4 ± 2.6 63.2 ± 25.9 1.6 ± 0.3 83.6 ± 3.7c 68 ± 2.3 60.3 ± 8.3 70.6 ± 5

T4 11.3 ± 1.7 25.7 ± 4.7 83.9 ± 10.5 0.39 ± 0.07 63.4 ± 3.1 57.3 ± 3.6 51.9 ± 3.4 74.9 ± 2.7 4.7 ± 0.5 6.3 ± 0.6 7.8 ± 1.1 75.3 ± 1.5* 72.6 ± 7.9 61.4 ± 3.2 T1 0.37 ± 0.07 75.9 ± 2.2 ** ** c ** b c b ** 0.19 ± 0.05 0.28 ± 0.07 0.11 ± 0.04 103.3 ± 4.2 115.9 ± 85.9 ± 10.2 T2 1.52 ± 0.5 62.9 ± 6.1 *** c *** c ** c *** c 13.8 *** c *** b HCP 228 ± 74 202 ± 59 426 ± 225 43.9 ± 5.6 T3 1.6 ± 0.3 40.5 ± 2.2* 37.6 ± 3.4* 66.9 ± 3 *** *** *** *** 44.7 ± 5.3 88.4 ± 21.1 T4 247 ± 24 ** 0.76 ± 0.14 51.1 ± 4.3* 43.7 ± 4* 37.7 ± 2.1* 67.7 ± 3.5 * * 13.4 ± 2.6 73.2 ± 2.6* 57.1 ± 1.1 T1 7.6 ± 0.8 ** 7.2 ± 1.3 ** 0.35 ± 0.08 66.9 ± 1.5 77.4 ± 1.3 ** c a 0.32 ± 0.05 0.28 ± 0.11 0.13 ± 0.03 93.2 ± 3.6 113.5 ± 93.1 ± 10.9 T2 1.46 ± 0.47 66.6 ± 3.9 *** c *** b *** c *** c 17.1 *** c *** c POL 307 ± 92 534 ± 225 44.8 ± 5.9 T3 189± 40 ** 2.1 ± 0.6 40.2 ± 1.1* 36.1 ± 4.5* 64.1 ± 1.8 *** *** *** 71.2 ± 15.2 115 ± 24.4 353 ± 52.7 T4 1.1 ±0.5 47.2 ± 4.1* 42.5 ± 5.6* 34.3 ±4.6 * 64.8 ± 2.9 ** ** *** T1 - one treatment by oral gavage 1 h before assays; T2 - three treatments during 24h (23, 5 and 1 h before sacrifice); T3 - two treatments per day during 5 days; T4 - two treatments per day during 5 days followed by a 3 days wash-out before assays. Data represent mean ± SEM from 4 separated experiments carried out in duplicate. Statistical difference calculated by one way ANOVA followed by Student-Newman-Keuls pairwise multiple comparison method. *p < 0.05; **p< 0.01; ***p< 0.001, significant difference in comparison to SAL in the same treatment regimen. ap < 0.05; bp< 0.01; cp< 0.001, significant difference in the same treatment group in comparison to others treatment regimen.

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In vitro experiments

In order to verify if the effects observed ex vivo were related to direct phloroglucinols action on receptor functionality, we evaluated the [35S]GTPγS binding stimulation by HC1 and HP4 in the absence of agonists. HC1 and HP4 were not able to stimulate the binding of [35S]GTPγS to any of the receptors in membranes prepared from non-treated animals (Figure 4.7).

100 A striata

90 hypothalamus frontal cortex 80 thalamus

on 70 ti

la 60 u m 50 ti S 40 % 30 20 10 0 -9 -8 -7 -6 log [HC1] (g/ml) 100 B 90 80

n 70 o i t

a

l 60 u m

i 50 t 40 %S 30

20 10

0 -9 -8 -7 -6 log [HP4] (g/ml)

Figure 4.7: Concentration-response effect of (A) HC1 and (B) HP4 on [35S]GTPγS binding. The data are presented as percentage of binding stimulation over GDP basal stimulation. Mean ± SEM from 4 separated experiments carried out in duplicate.

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4.5. DISCUSSION

The knowledge concerning alterations in receptor-G protein coupling in the depressive disorder (Chen et al., 1999; Gould and Manji, 2002; Donati and Rasenick, 2003), together with the findings that antidepressant agents may be effective on GPCR function (Hermans, 2003; Avissar and Schereiber, 2006) prompted us to investigate the effect H. caprifoliatum (HCP) and H. polyanthemum (POL) cyclohexane extracts on monaminergic and opioid receptors response in rat brain by measuring agonist-induced [35S]GTPγS binding. This was the first time that Hypericum species were evaluated regarding to post-synaptic biochemical responses after acute and repeated administration at doses they exert antidepressant-like activity in animal models.

Acute (T1) or three repeated treatment within 24h (T2) of rats with HCP or POL increased the binding of [35S]GTPγS to dopamine noradrenaline and serotonin receptors. In contrast five days treatment (T3) and five days with wash-out (T4) reduced the binding of 35 [ S]GTPγS. The EC50 values followed the same direction of Emax, T1 and T2 increased 35 monoamine potency to stimulate the [ S]GTPγS binding; T3 EC50 values were increased by 15 times or more, indicating a reduction on monoamine potency to stimulate the [35S]GTPγS binding, and even after 3 days wash-out (T4) this reduction was still present but in a lesser extent. Incubation of membranes with HC1 and HP4 without agonists did not influence [35S]GTPγS binding. Therefore the effects observed in the [35S]GTPγS binding after treating rats with POL and HCP could not be credited to agonist effect by HP4 or HC1 on monoaminergic receptors. Nevertheless, HC1 and HP4 inhibit monoamines uptake (Chapter 1 and 3), thus we speculate that the observed effects could be a consequence to monoamines increase in the synaptic cleft following the uptake inhibition. Hence, in T1 and T2, the increase in [35S]GTPγS binding could result from receptors adaptations intending to respond to the elevated levels of monoamines in the synapse. After 5 days treatment (T3 and T4), we can imagine that the decrease in [35S]GTPγS binding may be due to a desensitization or down regulation induced by the increased levels of monoamines in the synaptic clef (Stahl, 2000, Hermans, 2003; Spiegel, 2003). This result is in accordance with the majority

179 Confidencial 23/3/2007 experimental results that show a reduction in receptor-G protein coupling after chronic treatment with several classes of antidepressant drugs (Gould and Manji, 2002).

In fact, repeated exposure of a GPCR to agonist leads to a diminished responsiveness, that occurs through a variety of mechanisms, including desensitization, down-regulation and redistribution of cell surface receptors (trafficking) (Ferguson, 2001; Harrison and Traynor, 2003. Hermans, 2003). Donati and Rasenick (2003) point out several possible targets for antidepressant action via G proteins: (i) the number or affinity of receptors could be altered; receptors might show decreased affinity for agonist or they may be internalized; (ii) the ability of an agonist to activate a G protein might be decreased; (iii) the number of G proteins could be changed, p.ex. the activation of G protein genes or the stability of G protein at the cell surface could be affected. The intrinsic properties of a given G protein (e.g. affinity for GTP or rate of GTP hydrolysis) could be modified; (iv) the coupling between G proteins and their effectors could be altered, in this case, the ability of Gαs to activate (or Gαi to inhibit) adenylyl cyclase might be modified; (v) the effectors (p.ex. adenylyl cyclase) expression or activity could be altered by antidepressant treatment. Even though antidepressants’ effects on G protein functionality are variable, it appears that GTP-binding properties of G proteins are unaffected by antidepressant treatment (Emamghoreishi et al., 1996; Dwivedi and Pandey, 1997) and their effects on G protein signaling do not include changes in receptor or G protein gene expression (down regulation) (Hensler, 2002, 2003; Pejchal et al., 2002). Consequently, desensitization seems to play an important role in antidepressants effects on receptor-G protein coupling (Hermans, 2003; Spiegel, 2003).

Regarding the FST results, the effects of the extracts, HC1 and HP4 on [35S]GTPγS binding assay seem not to be directly related to antidepressant effects since T1, T2, T3 and T4 regimens were effective on forced swimming test. However, this observation does not necessarily mean that the antidepressant effect is not linked to the alterations observed in the [35S]GTPγS binding. It is known that FST is a test with good “predictive validity” but with low face and construct (Willner, 1990; Porsolt et al, 1991) this means that the model responds well to the same drugs as depressed humans even though its ability to simulate a depressive state is low. Accordingly, antidepressants that reduce immobility time acutely in the FST show

180 Confidencial 23/3/2007 their effects on GPCRs only after 14 -21 days of treatment (Hensler, 2002; Pejchal et al., 2002; Shen et al., 2002; Castro et al., 2003). Likewise, Galeotti et al. (2002) demonstrated that the inactivation of Gαi produces antiimmobility effect comparable to that produced by antidepressants, but this effect was observable only 11 days after pertussis toxin intracerebroventricular (i.c.v.) injection or 18-24h after antisense oligodeoxynucleotides directed against Gα (i.c.v.), while antidepressants were effective 30 minutes following treatment.

Our findings indicate that, at doses for which antidepressant-like activity have been demonstrated in animal models, H. caprifoliatum and H. polyanthemum extracts bring about adaptive changes in monoamine receptors. While within 24h the observed increase in [35S] GTPγS binding may result from receptors adaptations to cope with elevated levels of monoamines in the synapse. The reduced functional coupling of monoamine receptors to G proteins by 5 days treatment is in accord with the antidepressant mode of action. Furthermore, if the reduction in [35S] GTPγS binding after 5 days treatment with the extracts results from a desensitization process, this would explain the persistent antiimmobility observed after 3 days of wash-out.

Finally, the lack of influence over DAMGO-stimulated [35S]GTPγS binding curves stands for extracts selective effect on monoaminergic system. This result also fits with previous observation that, although the extracts present analgesic effects prevented by naloxone, their phloroglucinols do not inhibit [3H]naloxone binding (Chapter 1). From the [35S]GTPγS binding assay results we can infer that the cross-talk between monoaminergic and opioid systems would induce HCP and POL antinociceptive properties (Schereiber et al., 1998; Atkinson et al., 1999; Lynch, 2001), as already discussed in the Chapter 3.

In conclusion, the effects of H. caprifoliatum and H. polyanthemum on [35S]GTPγS confirms their prominent action on monoaminergic system and are consistent with antidepressants’ effects and may have some role in the antidepressant-like effect of these Hypericum species.

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III PARTE

DISCUSSÃO GERAL,

CONCLUSÕES E PERSPECTIVAS

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GENERAL DISCUSSION

Throughout this thesis, we carried out a study concerning to cyclohexane extracts of two Hypericum species native to South Brazil. Hypericum species have gained popularity for the treatment of mild to moderate depression (Linde et al., 1996; Butterweck et al., 1997; Müller et al., 1998; Müller, 2003). It has been shown that the antidepressant activity of Hypericum species would be due to several compounds such as naphthodianthrone, flavonoids, xanthones and phloroglucinol derivatives (Rocha et al., 1994; Chaterjee et al., 1998a; Butterweck et al., 2000). Studies, carried out by Chatterjee et al. (1998b) and Müller et al. (2001, 2003) groups, suggest that the Hypericum antidepressant-like effect would be assigned to the phloroglucinol derivatives, and more precisely to hyperforin. From these literature data and previous results obtained with Hypericum species native to Southern Brazil (Daudt et al., 2000; Gnerre et al., 2001; Viana 2002; Nör et al., 2004), the aim of our research was to investigate the potential antidepressant and analgesic effect of Brazilian Hypericum species, as well as to identify the substances responsible for these activities and to elucidate their mechanisms of action.

We have demonstrated that the antidepressant-like effect of H. caprifoliatum at rat FST, verified by Daudt et al. (2001) and Viana (2002) is reproducible, since it was also observed in mice and with different lipophilic extracts, i.e. petroleum ether and cyclohexane. We have also verified that H. polyanthemum cyclohexane extract (POL) has a pharmacological profile similar to that of H. caprifoliatum (HCP). Recent study carried out by Nor et al. (2004) revealed that phloroglucinol derivatives from Hypericum species, native to Southern Brazil have similar molecular pattern which differs from hyperforin and analogues obtained from H. perforatum. In spite of that, HC1, from H. caprifoliatum, and HP4, from H. polyanthemum, showed antidepressant like effect in the forced swimming test.

In order to investigate HCP and POL mechanism of action we performed pharmacological antagonism in vivo and neurochemical assays. In vivo pre-treatment with

SCH 23390 or sulpiride (D1 and D2 antagonists, respectively) abolished the anti-immobility effect of the extracts suggesting that the extracts antidepressant-like activity is associated with an effect on the monoaminergic systems. This assumption was reinforced by in vitro

188 Confidencial 23/3/2007 experiments in which the main phloroglucinol derivatives of H. caprifoliatum (HC1) and H. polyanthemum (HP4) dose dependently inhibited monoamine synaptosomal uptake, with more potency for dopamine, without binding to monoaminergic transporters. Our results are in agreement with the ones for other Hypericum species, H. perforatum (Chatterjee et al., 1998a; Müller et al., 1997, 1998) and H. triquetrifolium (Roz et al., 2002). These species also demonstrated antidepressant-like effect in the FST and monoamine uptake inhibition, related to the presence hyperforin, without affecting ligand specific binding to transporters (Chatterjee et al., 1998a, b; Müller et al., 1998, 2001; Jensen et al., 2001; Roz et al., 2002). Taken together, these latter observations and our results, strongly suggest that the antidepressant- like activity of Hypericum species is related to a mechanism different from classical antidepressants. However, the molecular mechanisms by which extracts of Hypericum species induce antidepressant-like activity, through monoamine uptake inhibition, remain unclear.

One hypothesis that could explain this effect is an alteration of monoamine transporters function or the reduction in the number of these transporters. Transporters are not quiescent plasma membrane proteins, plentiful evidence supports that they dynamically traffic to and from membrane, constitutively and in response to psychostimulant exposure, receptor activation and neuronal activity (do Rego, 2000; Chi and Reith, 2003; Melikian, 2004). Thus, incubation with HC1 and HP4 may reduce DA, NA and 5-HT uptake by reducing transporters in the membrane, this increases monoamines in the synaptic clef, but in a different way from psychostimulants, since they do not bind to the transporter. It is conceivable that DAT endocytosis occurs due to dopamine efflux as protective mechanism against dopamine metabolism that is known to be neurotoxic (Rabinovic et al., 2000).

The fact that Hypericum phloroglocinols derivatives influence monoamine uptake by a mechanism different from typical transporters inhibitors is coherent with their chemical structure. These molecules do not have nitrogen atom in their structure which is remarkably against structure activity relationship studies for monoamines transporters (Appell et al., 2004). Muller et coll have been searching alternative manners by which hyperforin influences neurotransmitters extracellular concentrations, firstly they found that it alters sodium

189 Confidencial 23/3/2007 conductive pathways (Singer et al., 1999) but the mechanism responsible for this effect remained uncertain. Recently, this group demonstrated that rats treated with hyperforin had the physico-chemical properties of brain neuronal membranes affected, increasing the flexibility of membrane acyl-chains and decreasing annular- as well as bulk-fluidity, which can be responsible for its pharmacological properties (Eckert et al., 2001, 2004).

It is a consensus among the field that the increase of monoamine levels in the synaptic clef caused by uptake inhibition is only the first from a series of neurochemical modifications induced by antidepressant. This increase evidently will lead to post synaptic modifications at different level of the signaling cascade (Stahl, 2000; Schreiber and Avissar, 2003; Millan, 2006). In addition, depressive patients present alterations in the hypothalamo-pituitary- adrenal (HPA) axis, like abnormal levels of corticosteroids, which are normalized by antidepressants; thus HPA axis has been considered as a target for antidepressant actions (Sachar et al., 1976; Gold et al., 1995; Holsboer and Barden, 1996; Nemeroff 1996; Pariante et al., 2004). For these reasons, we carried out the studies on receptor functionality through [35S]GTPγS binding assay and on corticosterone levels after different treatments regimens.

In relation to the effects on corticosterone levels, we demonstrated that only repeated treatment with antidepressants, imipramine and bupropion, or Hypericum extracts reduce forced swimming test induced corticosterone raise. Furthermore, differently from the antidepressants, HCP and POL decreased only cortical and not serum corticosterone levels. This result is in accordance with Franklin et al. (2004), who found decreased levels of corticosterone and cortisol after 14 days feeding rats with pellets containing H. perforatum extract. As discussed in Chapter 2, the authors suggest that this effect is related to induction of MDR P-glycoprotein, but the theory that antidepressant could restore the balance of HPA axis implies in the inhibition and not induction of this glycoprotein (Muller et al., 2003; Pariante et al., 2004; Pariante, 2006). Given that H. perforatum extracts and isolated substances, hypericin, hyperforin and flavonoids, are inducers of this glycoprotein at gastro-intestinal levels (Durr et al., 2000; Perloff et al., 2001; Hennessy et al., 2002; Webber et al., 2006), we consider more likely that extracts effect on HPA axis are via the monoaminergic activation follow-on uptake inhibition.

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Neverthless one should keep in mind that this set of experiments represents only a preliminary study of the Brazilian Hypericum species influence on the HPA axis functioning. Other parameters as CRH, POMC, ACTH, AVP, GR and MR should be evaluated in order to achieve more reliable conclusions.

Acutely the extracts increase both cortical and serum corticosterone levels in non- stressed mice, and although not significant, bupropion also has the same effect. Several studies report elevation in plasmatic HPA axis hormones in men and animals after acute or sub-chronic antidepressant treatment, p.ex. citalopram, fluoxetine, reboxetine, mirtazepine, H. perforatum extract WS 5570 and LI 160 (Seifritz et al., 1996; Duncan et al., 1998; Schule et al., 2004 a,b; Webber et al., 2006), and pergolide, a dopamine agonist (Fuller and Snoddy, 1984; Foreman et al., 1989). As discussed in Chapter 2, the monoamine elevation in the synaptic clef activates post-synaptic receptors in the hypothalamus and stimulates the secretion of several hormones (Raap and van de Kar 1999; Schule et al., 2004b). Moreover, one possible pathway involved glucocorticoid receptor (GR) resistance is the cAMP/PKA cascade, which is activated by monoamines (Pariante, 2006). There is now considerable evidence that phosphorylation of the GR by cAMP-dependent protein kinase has a relevant role in the regulation of GR function and depressed patients had been found to exhibit reduced cAMP-dependent protein kinase activity in cultured fibroblasts (Manier et al., 2000). Therefore, it is possible that disruption in the cAMP/PKA cascade described in major depression is linked to GR resistance in this disorder, and that antidepressants may overcome these receptor alterations via a direct effect on this pathway (Pariante, 2006). It should also be remembered that non-phosphortylated GR can physically associate to CREB, decreasing its CREB’s phosphorylation and thus reducing neuron survival (Holsboer, 2000), and neuronal death is one of the important features of the neurotrophic theory of depression (Duman et al., 1997). Major depression is associated with a selective loss of hippocampal volume that involves hypersecretion of glucocorticoids (Duman et al., 1997; Sapolsky, 2000).

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The results discussed until this point show that HCP, POL and their phloroglucinols derivatives have antidepressant effect in animal models comparable to tricyclics, bupropion and SSRI, but extracts’ neurochemical substrate is to some extent different from classic antidepressants. On the other hand, the results obtained after repeated treatments with the extracts in the [35S]GTPγS binding assay are in agreement with the majority of antidepressants chronic treatment studies, which demonstrate a decrease in receptor G- protein coupling (Gould and Manji, 2002) as consequence of a desensitization process (Hermans, 2003; Spiegel, 2003). As discussed in Chapter 4, the mechanism by which HCP and POL affects [35S]GTPγS binding induced by monoamines is not through direct binding of their phloroglucinols derivatives HC1 and HP4, since direct incubation with the phloroglucinols did not modify [35S]GTPγS binding. Differently from 5 days treatments (two treatments per days), acute (T1) and 3 treatments within 24h (T2) caused an increase in [35S]GTPγS binding induced by the three monoamines. Therefore, these divergent modifications according to the treatment regimen probably are adaptations to the elevation of monoamines in the synaptic clef resultant from uptake inhibition.

Considering the results by Eckert et al (2004) on membrane properties alterations after H. perforatum treatment, it is reasonable to propose that phloroglocinol derivatives, hyperforin, HC1 and uliginosin B, may behave in the plasma membrane similarly to cholesterol, modifying its ordering. Steroids modifications on membrane fluidity (Brann et al., 1995; Helmreich, 2003) can result in receptor or ion channel proteins conformational change and thereby influence its physiological function, for example neurotransmitter uptake, neurotransmitter synthesis, receptor binding and second messenger systems as well as signal transduction pathways (Clarke et al 1999, Helmreich, 2003). Membrane physico- chemical environment is important for G-protein activity, since these proteins are associated to the membrane and must move during the GTP cycle after agonist binding to the GPCRs (Donati and Rasenick, 2003; Helmreich, 2003; Hermans, 2003). It was demonstrated that cholesterol inclusion to the membrane matrix reduces its fluidity by restricting phospholipids mobility, hampering G-protein activation and so Gα association to adenylyl cyclase (Ropero et al., 2003).

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Although Hypericum phloroglocinols do not have the steroid skeleton, their massive prenylation renders the phloroglocinol nucleus highly lipophilic and makes it presumably apt to plunge in the plasma membrane. This could influence not only neurotransmitter uptake but also brain penetration of substances, as corticosterone. If this hypothesis is correct one may ask: why there is certain specificity for HC1 and uliginosin B to cause monoaminergic effects? It should be remembered that dopamine (DA), noradrenalin (NA) and serotonin (5-HT) belong to the same phylogenetic family (Figure 2) (Goldberg et al., 2003) and so they have more prone to be affected in the same way by modifications in their specific lipid environment.

Figure 2: Phylogenetic tree and predictions of the evolutionary model. (A) Phylogenetic tree of transporter proteins that were used to create the evolutionary model. This is a consensus tree from 2000 trees sampled during a Markov Chain Monte

Carlo run, using Mr. Bayes, state-of-the-art tree inference software. The tree is rooted at the midpoint of the longest branch. Transporters forming a monophyletic subfamily are indicated as, DA; dopamine transporters, NE; norepinephrine transporters, 5HT; serotonin transporters, GABA,

taurine, betaine, and creatine. The single sequence that does not fall under any of the subfamilies is an octopamine transporter from trichoplusia ni (cabbage looper) (SWISSPROT ID: Q95VZ4). Sequences at the end of branches indicated with 3 stars-Q22116, Q9V4E7, Q8TCC2 - are orphan

transporters from C. elegans, drosophila, and human, respectively. (From Goldber et al., 2002)

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Besides investigating the mechanism involved on the antidepressant-like effect of HCP and POL we also studied their analgesic activity. We can assume that extracts antinociceptive effect is likely to be related to the interaction between monoaminergic and opioid system. Although HCP and POL antinociceptive effect in the hot plate is prevented by naloxone, HC1 and uliginosin B did not affect [3H]-naloxone binding in membranes and the effect of DAMGO- induced [35S]GTPγS binding are not influenced when rats treated with the extracts. In addition, uliginosin B showed antinociceptive activity that was not prevented by naloxone pre-treatment. It is known that antidepressants have analgesic effect, mainly in chronic pain, without direct interaction binding to opioid receptors (Morgan and Franklin, 1991; Frussa-Filho et al., 1996; Altier and Stewart, 1999; Gilbert and Franklin, 2001). Moreover, morphine analgesia is increased by dopaminergic agonists and some of these agonists induce analgesia independently of opioid receptor (Morgan and Franklin, 1991; Altier and Stewart, 1998).

The results obtained for the extracts and phloroglucinol derivatives tested in this thesis as well as the extraction methods are summarized in Figure 3.

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Abbreviations: HCP, POL= cyclohexane extract from H. caprifoliatum and H. polyanthemum,

s

respectively t s i

n HC1, HP4 = phloroglucinol ed by DA ent

derivatives present in HCP ago ev r and POL, respectively ant CC= column P chromatography TLC = thin layer

⊕ l chromatography ng ⊕ a ng i = i

MA = monoamines m n m d om o

OPI = opioid i im t im i os w t b w test =

⊕ = positive result i

p te an g s h ⊕ a a d s n ⊕ l d n i

= negative result n Θ e e h c = t c t-p i t r

sy s Ho wr For test = take i Fo te MA p u

I P

m e A O Θ r 10 M tu :

= 1 exan s to rs o r

t = e g ed h L t to c-h 4 r : w en p 2 TLC n o v o CC l l y o HP4 PO HC1 o HCP 3X ce ndin b i sp fol ati prifolia re t:s B n an a r t acer pla M H. c H. polyanthemum

g Θ

n

i Θ = ⊕

d

ng e i : : c = d n

n one bin

i

ce r mi S ion = e Θ ⊕ t cal γ t

on i i ion by mi S b t t rti = γ os ula ed P ic Co ula t T im MA duc ressed OPI = r t t e s o S] GTP im r t s ress c 35 s n [ S] G St 35 [ No

Figure 3: Hypericum caprifoliatum and H. polyanthemum extraction scheme and pharmacological activities resume.

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Notably, this study corroborates with the idea that plant-derived substances have, and will certainly continue to have, a relevant place in the process of drug discovery (Calixto et al., 2000). Plants can be used as therapeutic resources in several ways: (i) herbal teas or other home made remedies; (ii) crude extracts or “standard enriched fractions” in phytopharmaceutical preparations or herbal medicines; (iii) subjected to successive extraction and purification procedures to isolate the compounds of interest, which can themselves be active and used directly as a drug, or as models for total synthesis, determining a prototype drug (Rates, 2001). Since item (iii) is the objective our work, we selected H. caprifoliatum and H. polyanthemum regarding their chemical content; the phloroglucinol derivatives, which have shown antidepressant like activity. We were able to demonstrate that H. caprifoliatum and H. polyanthemum and their phloroglucinol derivatives, HC1 and HP4, show antinociceptive effect and antidepressant-like activities in vivo and in vitro. Nevertheless, the mechanism by which these effects occur is different from the one of classical antidepressants. As discussed above, considering phloroglucinols molecular pattern and structural requirements to monoamine transporter binding, like presence of nitrogen atom, these results are not surprising (Appel et al., 2004).

Even so, phloroglucinols molecular peculiarities and lack of high selectivity for monoamine uptake do not prevent them from originating antidepressant drugs. Most experts agree that depression should be viewed as a syndrome, not a disease, since it affects various biological mechanisms and has a bidirectional link with co-morbidies (Charney and Manji, 2004; Evans et al., 2005; Berton and Nestler, 2006). In addition, drug discovery programs devoted in multi-target drugs strategies for treating depressive states and other complex CNS disorders, have been launched (Bymaster et al., 2001; for review see Millan, 2006). Drugs like the antidepressant tianeptine and the psychotropic olanzapine (used for the treatment of psychoses, schizophrenia and bipolar disorder) have their effect attributed to a complex, multitarget mechanism of action embracing modulation of monoaminergic, glutamatergic, and GABAergic networks, as well as other central acting systems (Bymaster et al., 2001; Millan, 2006). Natural products can be a source of substances with innovative mechanisms of action, still the challenges of developing and characterizing new drugs should not be neglected, and

196 Confidencial 23/3/2007 careful planned method for studying their extracts and isolated substances are decisive (Rates, 2001; Rollinger et al., 2006).

CONCLUSION AND PERSPECTIVES

The experimental results reported in this thesis provide evidences that plants remain a valuable source for new drugs search. We demonstrated that H. caprifoliatum (HCP) and H. polyanthemum (POL) cyclohexane extracts have potential antidepressant and analgesic activities. However they seem to have a pharmacological profile somewhat diverse from the classical antidepressant and opioid-like analgesics. Differently from tricyclics and monoamine selective reuptake inhibitors, the main extracts’ phloroglucinols, HC1 and HP4, inhibited dopamine ([3H]-DA), noradrenaline ([3H]-NA) and serotonin ([3H]-5HT) uptake but did not affect the binding of [3H]-mazindol, [3H]-nisoxetine and [3H]-citalopram to DA, NA and 5-HT transporters binding sites. Although the anti-immobility effect in the FST was inhibited by D1 35 and D2 antagonists and extracts treatment modify [ S]GTPγS binding profiles, HC1 and HP4 do not modify the [35S]GTPγS binding indicating that these phloroglucinols do not interact directly with monoaminergic receptors. Extracts’ effect on stress-induced corticosterone levels increase was also different from the antidepressants, while these drugs decrease cortical and serum corticosterone levels HCP and POL decreased only cortical levels. As well, the antinociceptive effect seems to be indirect. Naloxone prevented HCP and POL antinociceptive effect in the hot plate. Nonetheless, HC1 and HP4 did not inhibit [3H]-naloxone binding to opioid receptors, extracts treatment did no affect [35S]GTPγS binding nor HP4 antinociceptive effect was abolished by naloxone. In conclusion, H. caprifoliatum and H. polyanthemum analgesic and antidepressant effects engage the monoaminergic system and HPA axis, even though the manner extracts influence this system is different from classical antidepressants.

This conclusion is rather interesting since the research field for new antidepressants is also interested in new mechanisms of action. In order to substantiate the antidepressant potential HCP and POL will be tested on other animal models predictive of antidepressant

197 Confidencial 23/3/2007 action, like tail suspension test and learned helplessness, as well as on models with more construct validity, like depressed animals selective breeding. Several experiments will be clearly required to elucidate their mechanism of action. Microdialysis studies will be performed to verify extracts treatment effect on interstitial brain monoamine levels. Extracts’ effect on monoaminergic transporters and receptors number, after different treatment regimens, will be assessed by autoradiography binding. To refine the involvement of the monoaminergic system in the antinociceptive effect, pharmacological antagonism will be performed. In addition, the observation of some conflicting results between in vivo and in vitro assays as well as for extracts and isolated compounds make necessary a deeper chemical investigation and pharmacokinetic study since the effect at molecular level could be related to the minor substances or eventual metabolites. Finally, as the results clearly indicate that HCP and POL - and their main phloroglucinols HC1 and HP4 - activate the dopaminergic neurotransmission, the study of other illnesses related to the dopaminergic functions, such as Parkinson disease and substance abuse, is also planned for future investigations.

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SACHAR, E. J.; ROFFWARG, H. P.; GRUEN, P. H.; ALTMAN, N.; SASSIN, J. Neuroendocrine studies of depressive illness. Pharmakopsychiatrie, Neuro-Psychopharmakologie., v.9, p.11-17, 1976. SANCHEZ-MATEO, C. C.; PRADO, B.; RABANAL, R. M. Antidepressant effects of the methanol extracts of several Hypericum species from the Canary Islands. Journal of Ethnopharmacology, v.79, p.119-127, 2002. SANCHEZ-MATEO; C. C.; BONKANKA, C. X.;, HERNANDEZ-PEREZ, M.; RABANAL, R. M. Evaluation of the analgesic and topical anti-inflammatory effects of Hypericum reflexum L. fil. Journal of Ethnopharmacology, v.107, n.1, p.1-6, 2006. SAPOLSKY, R. M. The possibility of neurotoxicity in the hippocampus in major depression: a primer on neuron death. Biological Psychiatry, v. 48, p.755-765, 2000. SARRELL, E. M.; MANDELBERG, A.; COHEN, H. A. Efficacy of naturopathic extracts in the management of ear pain associated with acute otitis media. Archives of Pediatrics & Adolescent Medicine, v.155, n.7, p.796-799, 2001. SCHMITT, A. C. Detecção da atividade antiviral de plantas do gênero Hypericum (Guttiferae) nativas do Rio Grande do Sul sobre o vírus da imuno deficiência felina (FIV). Dissertação de Mestrado, UFRGS, 2000. 109p. SCHMITT , A. C.; RAVAZZOLO, A. P.; VON POSER , G.L. Investigation of some Hypericum species native to Southern of Brazil for antiviral activity. Journal of Ethnopharmacology, v. 77, p. 239–245, 2001. SCHREIBER, G.; AVISSAR, S. Application of G-proteins in the molecular diagnosis of psychiatric disorders. Expert review of molecular diagnostics, v.3, n.1, p.69-80, 2003. SCHULE, C.; BAGHAI, T.; SCHMIDBAUER, S.; BIDLINGMAIER, M.; STRASBURGER, C.J. LAAKMANN, G. Reboxetine acutely stimulates cortisol, ACTH, growth hormone and prolactin secretion in healthy male subjects. Psychoneuroendocrinology, v. 29, p.185-200, 2004b. SCHÜLE, C.; BAGHALI, T.; FERRERA, A.; LAAKMAN, G. Neuroendocrine effects of Hypericum extract WS 5570 in 12 healthy male volunteers. Pharmacopsychiatry, v.34, s. 1, p.127-133, 2001. SEIFRITZ, E.; BAUMANN, P.; MULLER, M.J.; ANNEN, O.; AMEY, M.; HEMMETER, U.; HATZINGER, M.; CHARDON, F.; HOLSBOER-TRACHSLER, E.. Neuroendocrine effects of a 20- mg citalopram infusion in healthy males. A placebo-controlled evaluation of citalopram as 5-HT function probe. Neuropsychopharmacology, v.14, p.253-263, 1996. SERKEDJIEVA, J.; MANOLOVA, N.; ZGORNIAK-NOWOSIELSKA, I.; ZAWILINSKA, B.; GRZYBEK, J. Antiviral activity of the infusion (SHS-174) from flowers of Sambucus nigra L., aerial parts of Hypericum perforatum L., and roots of Saponaria officinalis L. against influenza and herpes simplex viruses. Phytotherapy Research, v.4, n.3, p.97-100, 1990. SHELINE, Y.I.; SANGHAVI, M.; MINTUN, M. A.; GADO, M. H. Depression duration but not age predicts hippocampal volume loss in medically healthy women with recurrent major depression. Journal of Neuroscience, v.19, n.12, p.5034-5043, 1999. SHELTON, R.C.; KELLER, M.B.; GELENBERG, A.; DUNNER, D.L.; HIRSCHFELD, R.; THASE, M.E.; et al. Effectiveness of St John's Wort in major depression - A randomized controlled trial. Journal of the American Medical Association, v.285, n.15, p.1978-1986, 2001. SHIRAYAMA, Y.; CHEN, A. C.; NAKAGAWA, S.; RUSSELL, D. S.; DUMAN, R. S. Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. Journal of Neuroscience, v.22, n.8, p.3251-3261, 2002.

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SIMMEN, U.; BOBIRNAC, I.; ULMER, C.; LÜBBERT, H.; BÜTER, K. B.; SCHOEFFNER, W.; SCHOEFFTER, P. Antagonist effect of pseudohypericin at CRF1 receptors. European Journal of Pharmacology, v.458, p.251-256, 2003. SIMMEN, U.; HIGELIN, J.; BERGER-BÜTER, K.; SCHAFFNER, W.; LUNDSTROM, K. Neurochemical studies with St. John’s wort in vitro. Pharmacopsychiatry, v.34, s. 1, p. S137-S142, 2001. SINDRUP, S. H.; MADSEN, C.; BACH, F. W.; GRAM, L. F.; JENSEN, T. S. St. John's wort has no effect on pain in polyneuropathy. Pain, v. 91, p.361-365, 2000. SINGER, M. S.; WONNEMANN, M.; MÜLLER, W. E. Hyperforin, a major antidepressant constituint of St John's Wort, inhibits serotonin uptake by elevating free intracellular Na+1. The Journal of Pharmacology and Experimental Therapeutics, v.290, p.1363-1368, 1999. SINGH, Y. N. Potential for interaction of kava and St. John's wort with drugs. Journal of Ethnopharmacology, v.100, p.108-113, 2005. SIUCIAK, J. A.; LEWIS, D. R.; WIEGAND, S. J.; LINDSAY, R. M. Antidepressant-like effect of brain- derived neurotrophic factor (BDNF). Pharmacology Biochemistry and Behavior, v.56, pp. 131– 137, 1997. SKALISZ, L. L.; BEIJAMINI, V.; ANDREATINI, R. Effect of Hypericum perforatum on marble-burying by mice. Phytotherapy Research, v.18, p.5, p.399-402, 2004. SKALKOS, D.; STAVROPOULOS, N. E.; TSIMARIS, I.; GIOTI, E.; STALIKAS, C. D.; NSEYO, U. O.; IOACHIM, E.; AGNANTIS, N. J. The lipophilic extract of Hypericum perforatum exerts significant cytotoxic activity against T24 and NBT-II urinary bladder tumor cells. Planta Medica, v. 71, n.11, p.1030-1035, 2005. SPIEGEL, A. Cell signaling: beta-arrestin - not just for G protein-coupled receptors. Science, v.301, n.5638, p.1338-1339, 2003. STAFFELDT, B., KERB, R., BROCKMOLLER, J., PLOCH.; ROOTTS, I. Pharmacokinetics of hypericin and pseudohypericin after oral intake of the Hypericum perforatum extract LI 160 in healthy volunteers. Journal Geriatric Psyquiatry Neurology. v. 7, s.1, p. 47-53, 1994. STAHL, S. M. Essential Psychopharmacology: Neuroscientific Basis and Practical Applications. Cambridge: Cambridge university Press, 2nd ed, 2000. STAHL, S. M. Mechanism of action of serotonin selective reuptake inhibitors: Serotonin receptors and pathways mediate therapeutic effects and side effects. Journal of Affective Disorders, v.51, p.215–235, 1998. STEVINSON, C.; DIXON, M.; ERNST, E. Hypericum for fatigue – a pilot study. Phytomedicine, v.5, n.6, p.443-447, 1998. SUZUKI, O.; KATSUMATA, Y.; OYA, M.; BLADT, S.; WAGNER, H. Inhibition of monoamine oxidase by hipericin. Planta Medica, v. 50, p.272-4, 1984.

SZEGEDI, A.; KOHNEN, R.; DIENEL, A.; KIESER, M. Acute treatment of moderate to severe depression with hypericum extract WS 5570 (St John's wort): randomised controlled double blind non-inferiority trial versus paroxetine. British Medical Journal, v.330, p. 503-208, 2005. TADA, M.; CHIBA, K. YAMADA, H.; MARUYAMA, H. Phloroglucinol derivatives as competitive inhibitors against thrmboxane A2 and leukotriene D4 from Hyperium erectum. Phytochemistry, v. 30, n. 8, p. 2559-2562, 1991.;

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TAFET, G. E.; BERNARDINI, R. Psychoneuroendocrinological links between chronic stress and depression. Progress in Neuro-psychopharmacology and Biological Psychiatry, v. 27, p.893- 903, 2003. TAYLOR, R. S. L.; MANANDHAR, N. P.; HUDSON, J. B. and TOWERS, G. H. N. Antiviral activities of Nepalese medicinal plant. Journal of Ethnopharmacology, v.52, p. 157-163, 1996. THIEDE, H. M.; WALPER, A. Inhibition of MAO and COMT by Hypericum extracts and hypericin. Journal of Geriatric Psychiatry Neurology, v.7, s.1, p. 54-56, 1994. TIAN, R.; KOYABU, N.; MORIMOTO, S.; SHOYAMA, Y.; OHTANI, H.; SAWADA, Y. Functional induction and de-induction of P-glycoprotein by St. John's wort and its ingredients in a human colon adenocarcinoma cell line. Drug metabolism and disposition, v.33, n.4, p.547-54, 2005. TIRABOSCHI, E.; TARDITO, D.; KASAHARA, J.; MORASCHI, S.; PRUNERI, P.; GENNARELLI, M.; RACAGNI, G.; POPOLI, M. Selective Phosphorylation of Nuclear CREB by Fluoxetine is Linked to Activation of CaM Kinase IV and MAP Kinase Cascades. Neuropsychopharmacology, v.29, 1831–1840, 2004. TOKER, Z.; KIZIL, G.; OZEN, H. C.; KIZIL, M.; ERTEKIN, S. Compositions and antimicrobial activities of the essential oils of two Hypericum species from Turkey. Fitoterapia, v.77, n.1, p.57-60, 2006. TRIFUNOVIC, S.; VAJS, V.; MACURA, S.; JURANIC, N.; DJARMATI, Z.; JANKOV, R. AND MILOSAVLJEVIC, S. Oxidation products of hyperforin from Hypericum perforatum. Phytochemistry, v. 49, n. 5, p. 1305-1310, 1998. TROVATO, A.; RANERI, E.; KOULADIS, M.; TZAKOU, O.; TAVIANO, M. F.; GALATI, E. M. Anti- inflammatory and analgesic activity of Hypericum empertrifolium Willd. (Guttiferae). Il Farmaco, v.56, p.455-457, 2001. TSAI, S-J. TrkB partial agonists: Potential treatment strategy for major depression. Medical Hypotheses, doi:10.1016/j.mehy.2006.06.019, 2006. VANDENBOGAERDE, A.; GEBOES, K. R.; CUVEELE, J. F.; AGOSTINIS; P. M.; MERLEVEDE, W. J.; DE WITTE, P. A. Antitumour activity of photosensitized hypericin on A431 cell xenografts. Anticancer Research, v.16, n. 4A, p.1619-1625, 1996. VANDENBOGAERDE, A.; ZANOLI, P.; PUIA, G.; TRUZZI, C.; KAMUHABWA, A.; WITTE, P.; MERLEVEDE, W.; BARALDI, M. Evidence that total extract of Hypericum perforatum affects exploratory behavior and exerts anxiolitic effects in rats. Pharmacology Biochemistry and Behavior, v.65, n.4, p.627-633, 2000. VIANA, A. F.; ANDREAZZA, R. S.; DALLÁGNOL, R.; VON POSER, G. L.; RATES, S. R. K. Hypericum perforatum: elementos para o uso racional. Revista da AFARGS, encarte n. 9, 2001. VIANA, A. F. Estudo da atividade psicofarmacológica de espécies de Hypericum nativas do Rio Grande do Sul e toxicidade aguda de Hypericum caprifoliatum Cham. & Schledt. Dissertação de Mestrado, UFRGS, 2002. 124p VIANA A. F.; HECKLER A.P.; FENNER R.; RATES S.M.K. Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae). Brazilian Journal of Medical and Biological Research, v.36, p.631-634, 2003. VIANA A. F.; do REGO, J-C; VON POSER G.; FERRAZ A.; HECKLER A.P.; COSTENTIN J.; RATES S.M.K. The antidepressant like effect of Hypericum caprifoliatum Cham & Schlecht (Guttiferae) on forced swimming test results from an inhibition of neuronal monoamine uptake. Neuropharmacology, v.49, n. 7, p.1042-1052, 2005.

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VIANA, A.; DO REGO, J.C.; MUNARI, L.; DOURMAP, N.; HECKLER, A. P.; DALLA COSTA, T.; von POSER. G. L.; COSTENTIN, COSTENTIN, J.; RATES, S. M.K. Hypericum caprifoliatum (Guttiferae) Cham. & Schltdl.: a species native to South Brazil with antidepressant-like activity. Fundamental & Clinical Pharmacology, v.20, p.507–514, 2006. VIJAYAN, P.; RAGHU, C.; ASHOK, G.; DHANARAJ, S. A.; SURESH, B. Antiviral activity of medicinal plants of Nilgiris. The Indian journal of medical research, v.120, n.1, p.24-29, 2004. VOLZ, H. P. Controlled Clinical Trial of Hipericum Extracts in Depressed Pacients – an Overview. Pharmacopsychiatry, v.30, p.72-76, 1997. VORBACH, E.U.; HUBNER, W.D.; ARNOLDT, K.H. Effectiveness and tolerance of the Hypericum extracts L1 160 in comparison with imipramine: randomized double-blind study with 135 outpatients. Journal of Geriatric Psychiatry and Neurology, v.7 s.1, p.19-23. 1994 WAGNER, H.; BLADT, S. Pharmaceutical quality of Hypericum extracts. Journal of Geriatric Psychiatry and Neurology, v.7 s.1, p.65-68, 1994. WANG, E. J.; BARECKI-ROACH, M.; JOHNSON, W. W. Quantitative characterization of direct P- glycoprotein inhibition by St John's wort constituents hypericin and hyperforin. Journal of Pharmacy and Pharmacology, v.56, p.123-8, 2004. WEBER, C.C; ECKERT, G.P.; MULLER, W.E. Effects of antidepressants on the brain/plasma distribution of corticosterone. Neuropsychopharmacology, v.31, n.11, p.2443-2448, 2006. WHEATLEY, D. Safety of St John’s wort (Hypericum perforatum). Lancet, v.355, n.9203, p.575- 575, 2000 WHO, World Health Organization. 2006a. Disponível em http://www.searo.who.int/en/Section1174/Section1199/Section1567/Section1826_8097.htm. Acesso 25/out/2006 WHO, World Health Organization. 2006b Disponível em http://www.who.int/mediacentre/factsheets/fs265/en/. Acesso 25/out/2006 WILHELM, K. P.; BIEL, S.; SIEGERS, C. P. Role of flavonoids in controlling the phototoxicity of Hypericum perforatum . Phytomedicine , v.8, n.4, p.306-309, 2001. WINKELMANN, K.; HEILMANN, J.; ZERBE, O. New phloroglucinol derivatives from Hypericum papuanum. Journal of Natural Products, v.63, n.1, p.104-108, 2000. WINKELMANN, K.; HEILMANN, J.; ZERBE, O; RALI, T.; STICHER, O. New prenylated bi- and tricyclic phloroglucinol derivatives from Hypericum papuanum. Journal of Natural Products, v. 64, 701-706, 2001. WONNEMANN, M.; SINGER, A.; MÜLLER, W. E. Inhibition of sinaptossomal uptake of 3H-L- glutamate and 3H-GABA by hyperforin, a major constituent of St. John´s Wort: the role of amiloride sensitive sodium conductive pathways. Neuropsychopharmacology, v.23, n.2, p.188-197, 2000. WOOD, S. G.; HUFFMAN, J.; WEBER,N. D.; ANDERSEN, D.; NORTH, J. A.; MURRAY, B. K.; SIDWELL, R. and HUGHES, B. Antiviral activity of naturally occuring anthraquinones and anthraquinone derivatives. Planta Medica, v. 56, p. 651-652, 1990. YADID, G.; NAKASH,R.; DERI, I.; TAMAR,G.; KINOR, N.; GISPAN, I.; ZANGEN, A. Elucidation of the neurobiology of depression: insights from a novel genetic animal model. Progress in Neurobiology, v.62, p.353-378, 2000. YAMAKI, M.; ISHIGURO, K. Antimicrobial activity of naturally occurring and synthetic phloroglucinol against Staphylococcus aureus. Phytotherapy research, v.8, n. 2, p.112-114,1994.

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BIOGRAFIA

1 DADOS PESSOAIS

Nome: Alice Fialho Viana Filiação: Paulo Afonso Boução Viana e Terezinha de Jesus Fialho Viana Nascimento: 07/04/1975, Porto Alegre/ RS - Brasil

2 FORMAÇÃO ACADÊMICA/TITULAÇÃO

1994- 1998 Graduação em Farmácia. Universidade Federal do Rio Grande do Sul. 2000- 2002 Mestrado em Ciências Farmacêuticas. Programa de Pós-Graduação em Ciências farmacêuticas - UFRGS 2003- 2006 Doutorado em Ciências Farmacêuticas. Programa de Pós-Graduação em Ciências farmacêuticas - UFRGS e Université de Rouen

3 PRODUÇÃO CIENTÍFICA

3.1 Trabalhos apresentados

VIANA, Alice Fialho, COLVELLO, Ênio Vinícius; do REGO, Jean-Claude; COSTENTIN, Jean; RATES Stela Maris Kuze. Hypericum caprifoliatum effect on [35s]GTPγS binding to monoaminergic receptors and on forced swimming test. 30ª REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE FARMACOLOGIA TERAPÊUTICA E EXPERIMENTAL, 2006, Ribeirão Preto/SP, Brasil.

VIANA, Alice Fialho, JANIN, François, NAUDIN, Bertram, COSTENTIN, Jean; RATES Stela Maris Kuze. do REGO, Jean-Claude. Effect of acute or three days treatments by antidepressants on basal and stress-induced plasma and brain corticosterone levels. 10EME CONGRES DE LA SOCIETE FRANÇAISE DE PHARMACOLOGIE, 2006, Montpellier, France.

VIANA, Alice Fialho do REGO, Jean-Claude; HECKLER, Ana Paula; RATES Stela Maris Kuze; COSTENTIN, Jean. The involvement of monoamines in the antidepressant like activity of Hypericum caprifoliatum Cham & Schlecht (Guttiferae). 9EME CONGRES DE LA SOCIETE FRANÇAISE DE PHARMACOLOGIE, 2005, Bordeaux, France.

VIANA, Alice Fialho; do REGO, Jean-Claude; RATES Stela Maris Kuze; HECKLER, Ana Paula; COSTENTIN, Jean. The involvement of monoamines in the antidepressant like activity of Hypericum caprifoliatum Cham & Schlecht (Guttiferae). 8ÉME JOURNÉE SCIENTIFIQUE DU RÉSEAU LARC-NEUROSCIENCES, 2004, Paris, France.

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VIANA, Alice Fialho; RATES Stela Maris Kuze. HECKLER, Ana Paula; COSTENTIN, Jean, do REGO, Jean-Claude. Implication des monoamines dans les propriétés antidepressives potentielles de l’Hypericum polyanthemum. Journée de l’école doctorale Chimie-Biologie, 2005, Le Havre, France.

VIANA, Alice Fialho; do REGO, Jean-Claude; RATES Stela Maris Kuze; COSTENTIN, Jean. The mechanism of antidepressant like activity of Hypericum caprifoliatum Cham & Schlecht (Guttiferae) is mediated by the dopaminergic system. Journée de l’école doctorale Chimie-Biologie, 2004, Caen, France.

NEVES, Gilda; FENNER, Raquel; HECKLER, Ana Paula; VIANA, Alice Fialho; TASSO, Leandro; MENEGATTI, R; FRAGA, Carlos A M; BARREIRO, Eliezer Jesus; DALLACOSTA, Teresa Cristina; RATES, Stela M K. Evaluation of new n-phenylpiperazinic derivatives on dopaminergic mediated behaviors. XVII Reunião Anual da Federação de Sociedades de Biologia Experimental, 2002, Salvador, Brasil.

VIANA, Alice Fialho; HECKLER, Ana Paula; FENNER, Raquel; RATES, Stela Maris Kuze. Analgesic activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae). In: XVII Reunião Anual da Federação de Sociedades de Biologia Experimental, 2002, Salvador/BA, Brasil.

VIANA, Alice Fialho; BERNARDI, Ana Paula; FENNER, Raquel; SCHIMT, Aline; RAVAZOLO, Ana Paula; DRIEMEIER, David; POSER, Gilsane Lino Von; RATES, Stela Maris Kuze. Atividade antiviral e toxicidade in vitro e in vivo de espécies de Hypericum nativas do Rio Grande do Sul. In: X SIMPÓSIO LATINO AMERICANO E VII SIMPOSIO ARGENTINO DE FARMACOBOTÂNICA, 2001, Comodoro Rivadavia, Argentina.

VIANA, Alice Fialho; NEVES, Gilda Ângela; FENNER, Raquel; BERNARDI, Ana Paula; RATES, Stela Maris Kuze. Atividade dopaminérgica de Hypericum caprifoliatum Cham & Schlecht. In: IX JORNADS DE JOVENS PESQUISADORES DO GRUPO MONTEVIDEO, 2001, Rosário. 2001.

VIANA, Alice Fialho; NEVES, Gilda Ângela; PRIETSCH, Fernanda; FENNER, Raquel; GOSMANN, Grace; RATES, Stela Maris Kuze. Fracionamento bioguiado de Pfaffia glomerata Spreng (Amaranthaceae). In: XVI REUNIÃO ANUAL DA FEDERAÇÃO DE SOCIEDADES DE BIOLOGIA EXPERIMENTAL - FESBE, 2001, Caxambu/Mg, Brasil.

VIANA, Alice Fialho; FENNER, Raquel; BERNARDI, Ana Paula; SCHIMT, Aline; NEVES, Gilda Ângela; DRIEMEIER, David; POSER, Gilsane Lino Von; RATES, Stela Maris Kuze. Investigação da toxicidade aguda e atividades antidepressiva e ansiolítica de Hypericum caprifoliatum Cham & Schlecht em roedores. In: XVI REUNIÃO ANUAL DA FEDERAÇÃO DE SOCIEDADES DE BIOLOGIA EXPERIMENTAL - FESBE, 2001, Caxambu/MG, Brasil.

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GNERRE, Carmela; POSER, Gilsane Lino Von; VIANA, Alice Fialho; FERRAZ, Alexandre; TESTA, Bernard; RATES, Stela Maris Kuze. Monoamine Oxidase Inhibitory Activity of Some Hypericum Species Native to South Brazil. In: CONGRESS OF THE AMERICAS MEETING, 2001, Orlando / Florida, USA.

FENNER, Raquel; NEVES, Gilda Ângela; ALBRING, Daniela; VIANA, Alice Fialho; GNERRE, Carmela; BORDIGNON, Sérgio; POSER, Gilsane Lino Von; RATES, Stela Maris Kuze. Atividade Antidepressiva de espécies de Hypericum Nativas do Rio Grande do Sul. In: XII SALÃO DE INICIAÇÃO CIENTÍFICA-UFRGS, 2000, Porto Alegre/RS, Brasil.

VIANA, Alice Fialho; NEVES, Gilda Ângela; SÓRIA, Alessandra; DAUDT, Roberta; FERRAZ, Alexandre; BORDIGNON, Sérgio; POSER, Gilsane Lino Von; RATES, Stela Maris Kuze. Avaliação da atividade antidepressiva de espécies de Hypericum do Rio Grande do Sul. In: XI SALÃO DE INICIAÇÃO CIENTÍFICA, 2000, Porto Alegre/RS, Brasil.

RATES, Stela Maris Kuze; VIANA, Alice Fialho; FERRAZ, Alexandre; NEVES, Gilda Ângela; GNERRE, Carmela; POSER, Gilsane Lino Von; TESTA, Bernard. Atividade IMAO de espécies do gênero Hypericum (Guttiferae). In: XVI SIMPÓSIO DE PLANTAS MEDICINAIS DO BRASIL, 2000, Recife/PE, Brasil.

NEVES, Gilda Ângela; PRIETSCH, Fernanda; VIANA, Alice Fialho; FERRAZ, Alexandre; GNERRE, Carmela; SIMÕES, Cláudia M O; GOSMANN, Grace; TESTA, Bernard; RATES, Stela Maris Kuze. Screening farmacológico do extrato hidroalcoólico de Pfaffia glomerata Spreng (Amaranthaceae). In: XVI SIMPÓSIO DE PLANTAS MEDICINAIS DO BRASIL, 2000, Recife/ PE, Brasil.

VIANA, Alice Fialho; FENNER, Raquel; NEVES, Gilda Ângela; GNERRE, Carmela; ALBRING, Daniela; BORDIGNON, Sergio; POSER, Gilsane Lino Von; TESTA, Bernard; RATES, Stela Maris Kuze. Screening para a atividade antidepressiva de espécies de Hypericum nativas do Rio Grande do Sul. In: VIII JORNADS DE JOVENS PESQUISADORES DO GRUPO MONTEVIDEO, 2000, São Carlos /SP, Brasil.

VIANA, Alice Fialho; SÓRIA, Alessandra; FERRAZ, Alexandre; DAUDT, Roberta; BORDIGNON, Sérgio;POSER, Gilsane Lino Von; RATES, Stela Maris Kuze. Atividade antidepressiva e analgésica de Hypericum caprifoliatum Cham. & Schlet (Guttiferae). In: IX SIMPÓSIO LATINO-AMERICANO DE FARMACOBOTÂNCIA E III REUNIÃO LATINO- AMERICANA DE FITOQUÍMICA, 1999, Gramado/RS, Brasil.

VIANA, Alice Fialho; SORIA, Alessandra; NEVES, Gilda Ângela; FERRAZ, Alexandre; BORDIGNON, Sérgio; POSER, Gilsane Lino Von; RATES, Stela Maris Kuze. Avaliação da atividade antidepressiva e analgésica de Hypericum caprifoliatum Cham. & Schlecht (Guttiferae). In: XXV SEMANA ACADÊMICA DE ESTUDOS FARMACÊUTICOS (SAEF) - XX CONCURSO ACADÊMICO DE PESQUISA CIENTÍFICA- CAPEC, 1999, Porto Alegre/RS, Brasil.

VIANA, Alice Fialho; SÓRIA, Alessandra; FERRAZ, Alexandre; SCHMITT, Aline; STAATS, Charley; NEVES, Gilda Ângela; BORDIGNON, Sérgio; MANS, Dennis; SCHRIPSEMA, Jan;

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RAVAZZOLO, Ana Paula; RATES, Stela Maris Kuze; POSER, Gilsane Lino Von. Gênero Hypericum no Rio Grande do Sul (Brasil): fonte potencial de moléculas bioativas. In: VII JORNADAS DE JOVENS PESQUISADORES. CIÊNCIA PARA A PAZ, UNESCO/AUGM, 1999, Curitiba/PR, Brasil.

VIANA, Alice Fialho; POSER, Gilsane Lino Von; PÓLVORA, Jaqueline B. Plantas utilizadas nas religiões afro-brasileiras no Rio Grande do Sul. In: II SIMPÓSIO BRASILEIRO DE ETNOBIOLOGIA E ETNOFARMACOLOGIA, 1998, São Carlos/SP, Brasil.

BOEIRA, Jane M; VIANA, Alice Fialho; HENRIQUES, João Antônio Pegas. Citotoxic and Recombinogenic Effects of two b-carboline alkaloids in Saccharomyces cerevisiae. In: XXVI REUNIÃO ANUAL DA SBBQ, 1997, Caxambu/MG, Brasil.

3.2 Artigos completos publicados em periódicos

VIANA, Alice Fialho; do REGO, Jean-Claude; MUNARI, Leonardo; DOURMAP, Nathalie; Heckler, Ana Paula; DALLA COSTA, Teresa; von POSER. Gilsane L.; COSTENTIN, Jean ; Rates, Stela Maris Kuze. Hypericum caprifoliatum (Guttiferae) Cham. & Schltdl.: a species native to South Brazil with antidepressant-like activity. Fundamental & Clinical Pharmacology, v.20, p.507–514, 2006. do REGO, Jean-Claude; VIANA, Alice Fialho; LE MAITRE Erwan; DENIEL Audrey; RATES, Stela Maris Kuze; LEROUX-NICOLLET Isabelle, COSTENTIN, Jean. Comparisons between anxiety tests for selection of anxious and non anxious mice. Behavioural Brain Research, v. 169, p. 2, n. 282-288, 2006

VIANA, Alice Fialho; do REGO, Jean-Claude; von POSER Gilsane, FERRAZ, Alexandre; HECKLER, Ana Paula; COSTENTIN, Jean; RATES, Stela Maris Kuze. The antidepressant like effect of Hypericum caprifoliatum Cham & Schlecht (Guttiferae) on forced swimming test results from an inhibition of neuronal monoamine uptake. Neuropharmacology, v. 49, n. 7, p. 1042-1052, 2005.

VIANA, Alice Fialho ; HECKLER, Ana Paula; FENNER, Raquel; RATES, Stela Maris Kuze. Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae). Brazilian Journal of Medical and Biological Research, 36: 631-634, 2003.

NEVES, Gilda; FENNER, Raquel; HECKLER, Ana Paula; VIANA, Alice Fialho; TASSO, Leandro; MENEGATTI, Ricardo; FRAGA, Carlos Alberto; BARREIRO, Eliezer de Jesus, DALLA-COSTA Teresa; RATES, Stela Maris Kuze. Dopaminergic profile of new heterocyclic N-phenylpiperazine derivatives. Brazilian Journal of Medical Biological Research, 36: 625- 629, 2003.

VIANA, Alice Fialho; ANDREAZZA, Roberta; DALLAGNOL, Rodrigo; POSER, Gilsane Lino von; RATES, Stela Maris Kuze. Hypericum perforatum: elements for the rational use. Revista da AFARGS, v. 9, 2002.

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BOEIRA, Jane Marlei; VIANA, Alice Fialho; PICADA, Jaqueline; HENRIQUES, João Antônio. Genotoxic and recombinogenic activities of the two beta-carboline alkaloids harman and harmine in Saccharomyces cerevisiae. Mutatant Research, 20 (500): 39-48, 2002

GNERRE, Carmela; POSER, Gilsane Lino Von; FERRAZ, Alexandre; VIANA, Alice Fialho; TESTA, Bernard; RATES, Stela Maris Kuze. Monoamine oxidase inhibitory activity of some Hypericum species native to South Brazil. Journal of Pharmacy and Pharmacology, v. 53, n. 9, p. 1273-1279, 2001.

VIANA, Alice Fialho; ANDREAZZA, Roberte S; DALLAGNOL, Rodrigo; POSER, Gilsane Lino Von; RATES, Stela Maris Kuze. Hypericum perforatum: elementos para o uso racional. Revista da AFARGS, v. 9, 2001.

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